Polypeptides having Lysozyme Activity, Polynucleotides Encoding Same and Uses and Compositions Thereof

ABSTRACT

Animal feed or animal feed additives comprising one or more polypeptides having lysozyme activity. Polypeptides having lysozyme activity, polynucleotides encoding the polypeptides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to novel LYS polypeptides having lysozymeactivity, polynucleotides encoding the polypeptides, nucleic acidconstructs, vectors, and host cells comprising the polynucleotides aswell as methods of producing the polypeptides. The present inventionalso relates to compositions, specifically animal feed, comprising LYSpolypeptides and the use of the LYS polypeptide in animal feed.

Description of the Related Art

Lysozyme is an O-glycosyl hydrolase produced as a defensive mechanismagainst bacteria by many organisms. The enzyme causes the hydrolysis ofbacterial cell walls by cleaving the glycosidic bonds of peptidoglycan;an important structural molecule in bacteria. After having their cellwalls weakened by lysozyme action, bacterial cells lyse as a result ofunbalanced osmotic pressure.

Lysozyme naturally occurs in many organisms such as viruses, plants,insects, birds, reptiles and mammals. In mammals, Lysozyme has beenisolated from nasal secretions, saliva, tears, intestinal content, urineand milk. The enzyme cleaves the glycosidic bond between carbon number 1of N-acetylmuramic acid and carbon number 4 of N-acetyl-D-glucosamine.In vivo, these two carbohydrates are polymerized to form the cell wallpolysaccharide of many microorganisms.

Lysozyme has until now been classified into seven different glycosidehydrolase (GH) families (CAZy, www.cazy.org): GH18, GH19, hen egg-whitelysozyme (GH22), goose egg-white lysozyme (GH23), bacteriophage T4lysozyme (GH24), Sphingomonas flagellar protein (GH73) and Chalaropsislysozymes (GH25).

Lysozyme extracted from hen egg white is the primary product availableon the commercial market, but does not cleave N,6-O-diacetylmuramic acidin e.g. Staphylococcus aureus cell walls and is thus unable to lyse thisimportant human pathogen among others (Masschalck B, Deckers D, MichielsCW (2002), “Lytic and nonlytic mechanism of inactivation ofgram-positive bacteria by lysozyme under atmospheric and highhydrostatic pressure”, J Food Prot. 65(12):1916-23).

Use of lysozyme has been suggested in animal feed (see for example WO00/21381 and WO 04/026334), in cheese production (see for example WO05/080559), food preservation (Hughey and Johnson (1987) Appl EnvironMicrobiol 53:2165), detergents (see for example U.S. Pat. No. 5,041,236and EP 0425016), in oral care (see for example U.S. Pat. No. 4,355,022,WO 04/017988 and WO 08/124764), cosmetology and dermatology,contraception, urology, and gynaecology (see for example WO 08/124764).

Antimicrobial growth promoters (AGP's) have traditionally been used forgrowth promotion in animals, and probably work by preventing low levelinfections by pathogens such as Clostridium perfringens. However, AGP'sare increasingly being banned worldwide and therefore new solutions topromote animal growth but which are not AGP's are of interest.

SUMMARY OF THE INVENTION

The inventors have discovered a completely novel class of polypeptideshaving lysozyme activity. As such, the invention relates to acomposition comprising at least 0.01 mg of LYS polypeptide per kilogramof composition, wherein the polypeptide (a) has lysozyme activity and(b) comprises one or more LAD catalytic domains; wherein the LADcatalytic domain gives a domT score of at least 180 when queried using aProfile Hidden Markov Model (HMM) prepared using SEQ ID NOs: 46 to 187and hmmbuild software program. Typically, the query is carried out usinghmmscan software program by the Method of Determining the LAD CatalyticDomain by HMM.

The invention further relates to an isolated polypeptide having lysozymeactivity, selected from the group consisting of:

-   -   (a) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 3;    -   (b) a polypeptide having at least 94% sequence identity to the        polypeptide of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 12;    -   (e) a polypeptide having at least 87% sequence identity to the        polypeptide of SEQ ID NO: 15;    -   (f) a polypeptide having at least 81% sequence identity to the        polypeptide of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 24;    -   (i) a polypeptide having at least 87% sequence identity to the        polypeptide of SEQ ID NO: 27;    -   (j) a polypeptide having at least 96.2% sequence identity to the        polypeptide of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 36;    -   (m) a polypeptide having at least 81% sequence identity to the        polypeptide of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 42;    -   (o) a variant of the polypeptide of SEQ ID NO: 3, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 positions;    -   (p) a variant of the polypeptide of SEQ ID NO: 6, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13        positions;    -   (q) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 9 and SEQ ID NO: 36, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 positions;    -   (r) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 24, SEQ        ID NO: 33 and SEQ ID NO: 42, wherein the variant has lysozyme        activity and comprises one or more amino acid substitutions,        and/or one or more amino acid deletions, and/or one or more        amino acid insertions or any combination thereof in 1, 2, 3, 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,        38, 39, 40, 41, 42, 43, 44 or 45 positions;    -   (s) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 15 and SEQ ID NO: 27, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29        positions;    -   (t) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 18 and SEQ ID NO: 39, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 positions;    -   (u) a variant of the polypeptide of SEQ ID NO: 30, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7 or 8 positions;    -   (v) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p),        (q), (r), (s), (t) or (u) and a N-terminal and/or C-terminal        His-tag and/or HQ-tag;    -   (w) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p),        (q), (r), (s), (t) or (u) and a N-terminal and/or C-terminal        extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8,        9 or 10 amino acids; and    -   (x) a fragment of the polypeptide of (a), (b), (c), (d), (e),        (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r),        (s), (t) or (u) having lysozyme activity and having at least 90%        of the length of the mature polypeptide.

The invention also relates to animal feed additives or animal feedcomprising the LYS polypeptide of the invention; use of the lysozyme ofthe LYS polypeptide in animal feed, in animal feed additives, in thepreparation of a composition for use in animal feed, and for improvingone or more performance parameters in an animal. The invention furtherrelates to methods of improving performance parameters of an animal andfor preparing an animal feed; isolated polynucleotides encoding thepolypeptides of the invention, nucleic acid constructs, recombinantexpression vectors, recombinant host cells and method of producing theLYS polypeptide of the invention. The invention is further directed tothe use of composition of of the invention in animal feed; in animalfeed additives; in the preparation of a composition for use in animalfeed; for improving the nutritional value of an animal feed; forincreasing digestibility of the animal feed; and/or for improving one ormore performance parameters in an animal.

Overview of Sequence Listing

SEQ ID NO: 1 is the cDNA sequence of a LYS polypeptide as isolated fromPenicillium simplicissimum.

SEQ ID NO: 2 is the amino acid sequence as deduced from SEQ ID NO: 1.

SEQ ID NO: 3 is the amino acid sequence of the mature LYS polypeptidefrom Penicillium simplicissimum.

SEQ ID NO: 4 is the cDNA sequence of a LYS polypeptide as isolated fromPenicillium vasconiae.

SEQ ID NO: 5 is the amino acid sequence as deduced from SEQ ID NO: 4.

SEQ ID NO: 6 is the amino acid sequence of the mature LYS polypeptidefrom Penicillium vasconiae.

SEQ ID NO: 7 is the cDNA sequence of a LYS polypeptide as isolated fromTalaromyces proteolyticus.

SEQ ID NO: 8 is the amino acid sequence as deduced from SEQ ID NO: 7.

SEQ ID NO: 9 is the amino acid sequence of the mature LYS polypeptidefrom Talaromyces proteolyticus.

SEQ ID NO: 10 is the cDNA sequence of a LYS polypeptide as isolated fromAspergillus sp. XZ2668.

SEQ ID NO: 11 is the amino acid sequence as deduced from SEQ ID NO: 10.

SEQ ID NO: 12 is the amino acid sequence of the mature LYS polypeptidefrom Aspergillus sp. XZ2668.

SEQ ID NO: 13 is the cDNA sequence of a LYS polypeptide as isolated fromPenicillium antarcticum.

SEQ ID NO: 14 is the amino acid sequence as deduced from SEQ ID NO: 13.

SEQ ID NO: 15 is the amino acid sequence of the mature LYS polypeptidefrom Penicillium antarcticum.

SEQ ID NO: 16 is the cDNA sequence of a LYS polypeptide as isolated fromOvatospora brasiliensis.

SEQ ID NO: 17 is the amino acid sequence as deduced from SEQ ID NO: 16.

SEQ ID NO: 18 is the amino acid sequence of the mature LYS polypeptidefrom Ovatospora brasiliensis.

SEQ ID NO: 19 is the cDNA sequence of a LYS polypeptide as isolated fromPenicillium wellingtonense.

SEQ ID NO: 20 is the amino acid sequence as deduced from SEQ ID NO: 19.

SEQ ID NO: 21 is the amino acid sequence of the mature LYS polypeptidefrom Penicillium wellingtonense.

SEQ ID NO: 22 is the cDNA sequence of a LYS polypeptide as isolated fromPenicillium roseopurpureum.

SEQ ID NO: 23 is the amino acid sequence as deduced from SEQ ID NO: 22.

SEQ ID NO: 24 is the amino acid sequence of the mature LYS polypeptidefrom Penicillium roseopurpureum.

SEQ ID NO: 25 is the cDNA sequence of a LYS polypeptide as isolated fromPenicillium virgatum.

SEQ ID NO: 26 is the amino acid sequence as deduced from SEQ ID NO: 25.

SEQ ID NO: 27 is the amino acid sequence of the mature LYS polypeptidefrom Penicillium virgatum.

SEQ ID NO: 28 is the cDNA sequence of a LYS polypeptide as isolated fromAspergillus niveus.

SEQ ID NO: 29 is the amino acid sequence as deduced from SEQ ID NO: 28.

SEQ ID NO: 30 is the amino acid sequence of the mature LYS polypeptidefrom Aspergillus niveus.

SEQ ID NO: 31 is the cDNA sequence of a LYS polypeptide as isolated fromChaetomium sp. ZY369.

SEQ ID NO: 32 is the amino acid sequence as deduced from SEQ ID NO: 31.

SEQ ID NO: 33 is the amino acid sequence of the mature LYS polypeptidefrom Chaetomium sp. ZY369.

SEQ ID NO: 34 is the cDNA sequence of a LYS polypeptide as isolated fromTalaromyces atricola.

SEQ ID NO: 35 is the amino acid sequence as deduced from SEQ ID NO: 34.

SEQ ID NO: 36 is the amino acid sequence of the mature LYS polypeptidefrom Talaromyces atricola.

SEQ ID NO: 37 is the cDNA sequence of a LYS polypeptide as isolated fromTrichocladium asperum.

SEQ ID NO: 38 is the amino acid sequence as deduced from SEQ ID NO: 37.

SEQ ID NO: 39 is the amino acid sequence of the mature LYS polypeptidefrom Trichocladium asperum.

SEQ ID NO: 40 is the cDNA sequence of a LYS polypeptide as isolated fromMetarhizium carneum.

SEQ ID NO: 41 is the amino acid sequence as deduced from SEQ ID NO: 40.

SEQ ID NO: 42 is the amino acid sequence of the mature LYS polypeptidefrom Metarhizium carneum.

SEQ ID NO: 43 is the cDNA sequence of a LYS polypeptide as isolated fromThielavia terrestris.

SEQ ID NO: 44 is the amino acid sequence as deduced from SEQ ID NO: 43.

SEQ ID NO: 45 is the amino acid sequence of the mature LYS polypeptidefrom Thielavia terrestris.

SEQ ID NO: 46 is the amino acid sequence of the LAD domain ofSWISSPROT:A1C4L9 from Aspergillus clavatus.

SEQ ID NO: 47 is the amino acid sequence of the LAD domain ofSWISSPROT:A4X6S9 from Salinispora tropica.

SEQ ID NO: 48 is the amino acid sequence of the LAD domain ofSWISSPROT:A8M1H3 from Salinispora arenicola.

SEQ ID NO: 49 is the amino acid sequence of the LAD domain ofSWISSPROT:Q3L9Z6 from Rhodococcus erythropolis.

SEQ ID NO: 50 is the amino acid sequence of the LAD domain ofSWISSPROT:B5U576 from Mycobacterium phage Pacc40.

SEQ ID NO: 51 is the amino acid sequence of the LAD domain ofSWISSPROT:B6GZX8 from Penicillium rubens.

SEQ ID NO: 52 is the amino acid sequence of the LAD domain ofSWISSPROT:D1S6X5 from Micromonospora aurantiaca.

SEQ ID NO: 53 is the amino acid sequence of the LAD domain ofSWISSPROT:D1S8J3 from Micromonospora aurantiaca.

SEQ ID NO: 54 is the amino acid sequence of the LAD domain ofSWISSPROT:D1SH66 from Micromonospora aurantiaca.

SEQ ID NO: 55 is the amino acid sequence of the LAD domain ofSWISSPROT:D5GBH0 from Tuber melanosporum.

SEQ ID NO: 56 is the amino acid sequence of the LAD domain ofSWISSPROT:G9P583 from Hypocrea atroviridis.

SEQ ID NO: 57 is the amino acid sequence of the LAD domain ofSWISSPROT:E9ED38 from Metarhizium acridum.

SEQ ID NO: 58 is the amino acid sequence of the LAD domain ofSWISSPROT:E9FAK9 from Metarhizium robertsii.

SEQ ID NO: 59 is the amino acid sequence of the LAD domain ofSWISSPROT:F4F8N8 from Verrucosispora maris.

SEQ ID NO: 60 is the amino acid sequence of the LAD domain ofSWISSPROT:F4F159 from Verrucosispora maris.

SEQ ID NO: 61 is the amino acid sequence of the LAD domain ofSWISSPROT:J4USU4 from Beauveria bassiana.

SEQ ID NO: 62 is the amino acid sequence of the LAD domain ofSWISSPROT:G2QV10 from Thielavia terrestris.

SEQ ID NO: 63 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6W456 from Micromonospora peucetia.

SEQ ID NO: 64 is the amino acid sequence of the LAD domain ofSWISSPROT:H8E7T0 from Microbacterium laevaniformans.

SEQ ID NO: 65 is the amino acid sequence of the LAD domain ofSWISSPROT:I0PF45 from Mycobacterium abscessus.

SEQ ID NO: 66 is the amino acid sequence of the LAD domain ofSWISSPROT:10L0M9 from Micromonospora lupini str Lupac.

SEQ ID NO: 67 is the amino acid sequence of the LAD domain ofSWISSPROT:I0L3A4 from Micromonospora lupini str Lupac.

SEQ ID NO: 68 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0B2X541 from Metarhizium album.

SEQ ID NO: 69 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A168BM L7 from Aschersonia aleyrodis.

SEQ ID NO: 70 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0B2WV75 from Metarhizium album.

SEQ ID NO: 71 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A167BVWV4 from Cordyceps brongniartii.

SEQ ID NO: 72 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A167ECQ5 from Metarhizium rileyi.

SEQ ID NO: 73 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A162JZ16 from Cordyceps confragosa.

SEQ ID NO: 74 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A168DNP6 from Cordyceps confragosa.

SEQ ID NO: 75 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A168DOL5 from Cordyceps confragosa.

SEQ ID NO: 76 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A168BQC6 from Isaria fumosorosea.

SEQ ID NO: 77 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A167X055 from Isaria fumosorosea.

SEQ ID NO: 78 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A167NNI6 from Isaria fumosorosea.

SEQ ID NO: 79 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A179H6H8 from Purpureociffium lilacinum.

SEQ ID NO: 80 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A179FH10 from Pochonia chlamydosporia.

SEQ ID NO: 81 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A179F665 from Pochonia chlamydosporia.

SEQ ID NO: 82 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A179F1Q1 from Pochonia chlamydosporia.

SEQ ID NO: 83 is the amino acid sequence of the LAD domain ofSWISSPROT:S7ZNE7 from Penicillium oxalicum.

SEQ ID NO: 84 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0D9PBV5 from Metarhizium anisopliae.

SEQ ID NO: 85 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0D9NPP1 from Metarhizium anisopliae.

SEQ ID NO: 86 is the amino acid sequence of the LAD domain ofSWISSPROT:W6QNL2 from Penicillium roqueforti.

SEQ ID NO: 87 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0U1M0W5 from Talaromyces islandicus.

SEQ ID NO: 88 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0A219P6 from Penicillium expansum.

SEQ ID NO: 89 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A086T4C8 from Acremonium chrysogenum.

SEQ ID NO: 90 is the amino acid sequence of the LAD domain ofSWISSPROT:X8ERY9 from Mycobacterium chelonae.

SEQ ID NO: 91 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A081HTU5 from Mycobacterium sp TKK.

SEQ ID NO: 92 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0A1TMJ0 from Torrubiella hemipterigena.

SEQ ID NO: 93 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0A1TNZ8 from Torrubiella hemipterigena.

SEQ ID NO: 94 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0A1D149 from Arthrobacter sp PAMC.

SEQ ID NO: 95 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0A6UNI9 from Actinoplanes utahensis.

SEQ ID NO: 96 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0B4I0X1 from Metarhizium majus.

SEQ ID NO: 97 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0D0WU99 from Micromonospora carbonacea.

SEQ ID NO: 98 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0D1LTE6 from Mycobacterium immunogenum.

SEQ ID NO: 99 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0F8A5E8 from Hirsutella minnesotensis.

SEQ ID NO: 100 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0F8A617 from Hirsutella minnesotensis.

SEQ ID NO: 101 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0F7TVL0 from Penicillium brasilianum.

SEQ ID NO: 102 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0L0N1U6 from Tolypocladium ophioglossoides.

SEQ ID NO: 103 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0M0UGY1 from Madurella mycetomatis.

SEQ ID NO: 104 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0K8L1J1 from Aspergillus udagawae.

SEQ ID NO: 105 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0H5NX60 from Nocardia farcinica.

SEQ ID NO: 106 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0M2RBW0 from Micromonospora sp HK10.

SEQ ID NO: 107 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0M2RKI6 from Micromonospora sp HK10.

SEQ ID NO: 108 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1F5LVD8 from Penicillium murcianum.

SEQ ID NO: 109 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0M8XNG9 from Micromonospora sp.

SEQ ID NO: 110 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0W7W0M4 from Trichoderma gamsii.

SEQ ID NO: 111 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0Q9MHJ4 from Arthrobacter sp Soil761.

SEQ ID NO: 112 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0Q9MU26 from Arthrobacter sp Soil736.

SEQ ID NO: 113 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0P0DUT5 from Microbacterium sp No 7.

SEQ ID NO: 114 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0Q9N9Z1 from Arthrobacter sp Soil762.

SEQ ID NO: 115 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6RVB7 from Micromonospora halophytica.

SEQ ID NO: 116 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6SUA9 from Micromonospora nigra.

SEQ ID NO: 117 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A135LMU8 from Penicillium patulum.

SEQ ID NO: 118 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0S9BYR1 from Arthrobacter sp Lea169.

SEQ ID NO: 119 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0U0ZSQ6 from Mycobacterium abscessus.

SEQ ID NO: 120 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A100WIQ1 from Mycobacterium canariasense.

SEQ ID NO: 121 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1091P50 from Micromonospora rifamycinica.

SEQ ID NO: 122 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A109IHN3 from Micromonospora rifamycinica.

SEQ ID NO: 123 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A0S2M353 from Arthrobacter alpinus.

SEQ ID NO: 124 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A134DEL4 from Microbacterium hominis.

SEQ ID NO: 125 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A142KAG2 from Gordonia phage Obliviate.

SEQ ID NO: 126 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A136PN50 from Micromonospora rosaria.

SEQ ID NO: 127 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A138A7X6 from Tsukamurella pseudospumae.

SEQ ID NO: 128 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A136PTZ6 from Micromonospora rosaria.

SEQ ID NO: 129 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A177U5Z0 from Tilletia walkeri.

SEQ ID NO: 130 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A177VGU0 from Tilletia controversa.

SEQ ID NO: 131 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A179G202 from Pochonia chlamydosporia 170.

SEQ ID NO: 132 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A179G1N9 from Pochonia chlamydosporia 170.

SEQ ID NO: 133 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A179FEB3 from Pochonia chlamydosporia 170.

SEQ ID NO: 134 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A179HTK7 from Purpureocillium lilacinum.

SEQ ID NO: 135 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1770655 from Paraphaeosphaeria sporulosa.

SEQ ID NO: 136 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A167GE76 from Cordyceps brongniartii RCEF 3172.

SEQ ID NO: 137 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A160DID3 from Gordonia phage Utz.

SEQ ID NO: 138 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A175J866 from Arthrobacter nicotinovorans.

SEQ ID NO: 139 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1A1X5E5 from Mycobacterium conceptionense.

SEQ ID NO: 140 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1A8ZCI7 from Micromonospora narathiwatensis.

SEQ ID NO: 141 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1A8Z6H4 from Micromonospora narathiwatensis.

SEQ ID NO: 142 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1A8Z6S5 from Micromonospora auratinigra.

SEQ ID NO: 143 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1A2MHG1 from Mycobacterium sp E1747.

SEQ ID NO: 144 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1A9BD29 from Micromonospora sediminicola.

SEQ ID NO: 145 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A196L8B1 from Microbacterium sp H83.

SEQ ID NO: 146 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C4X8A3 from Micromonospora coriariae.

SEQ ID NO: 147 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6U2J5 from Micromonospora citrea.

SEQ ID NO: 148 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6V2H5 from Micromonospora peucetia.

SEQ ID NO: 149 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6VFJ6 from Micromonospora yangpuensis.

SEQ ID NO: 150 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6S481 from Micromonospora rhizosphaerae.

SEQ ID NO: 151 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C5K5N0 from Micromonospora echinaurantiaca.

SEQ ID NO: 152 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C5K0N2 from Micromonospora inositola.

SEQ ID NO: 153 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C5JUR0 from Micromonospora inositola.

SEQ ID NO: 154 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C5JX99 from Micromonospora coxensis.

SEQ ID NO: 155 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C4ZAM5 from Micromonospora mirobrigensis.

SEQ ID NO: 156 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C4Z5B4 from Micromonospora viridifaciens.

SEQ ID NO: 157 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C4YQ99 from Micromonospora haikouensis.

SEQ ID NO: 158 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6W5T7 from Micromonospora peucetia.

SEQ ID NO: 159 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6W1B9 from Micromonospora citrea.

SEQ ID NO: 160 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C5A7S5 from Micromonospora saelicesensis.

SEQ ID NO: 161 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C5A2Q7 from Micromonospora echinospora.

SEQ ID NO: 162 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C4ZJ35 from Micromonospora purpureochromogenes.

SEQ ID NO: 163 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C5G758 from Micromonospora echinofusca.

SEQ ID NO: 164 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C5H5B2 from Micromonospora echinaurantiaca.

SEQ ID NO: 165 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6TPW2 from Micromonospora citrea.

SEQ ID NO: 166 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C5IVI4 from Micromonospora echinaurantiaca.

SEQ ID NO: 167 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6SECO from Micromonospora pallida.

SEQ ID NO: 168 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6SF84 from Micromonospora rhizosphaerae.

SEQ ID NO: 169 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C6SQT8 from Micromonospora pallida.

SEQ ID NO: 170 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C3N2H1 from Micromonospora krabiensis.

SEQ ID NO: 171 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C3N0X1 from Micromonospora krabiensis.

SEQ ID NO: 172 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C9EHM2 from Mycobacterium phage Tonenili.

SEQ ID NO: 173 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1C9EHF6 from Mycobacterium phage Tonenili.

SEQ ID NO: 174 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1E41B54 from Pseudonocardia sp SCN 72-86.

SEQ ID NO: 175 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1G8AK84 from Microbacterium pygmaeum.

SEQ ID NO: 176 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1E4NTZ4 from Pseudonocardia sp SCN 73-27.

SEQ ID NO: 177 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1J0MA43 from Mycobacterium phage Lukilu.

SEQ ID NO: 178 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1N6R3C0 from Micromonospora avicenniae.

SEQ ID NO: 179 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1N0VNN7 from Mycobacterium abscessus subsp abscessus.

SEQ ID NO: 180 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1N4DHL3 from Mycobacterium abscessus subsp abscessus.

SEQ ID NO: 181 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1N4RVY0 from Mycobacterium abscessus subsp abscessus.

SEQ ID NO: 182 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1N6X5P6 from Micromonospora avicenniae.

SEQ ID NO: 183 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1N2VK68 from Mycobacterium abscessus subsp abscessus.

SEQ ID NO: 184 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1N1GK82 from Mycobacterium abscessus subsp abscessus.

SEQ ID NO: 185 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1N1EP78 from Mycobacterium abscessus subsp abscessus.

SEQ ID NO: 186 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1N1G9F3 from Mycobacterium abscessus subsp abscessus.

SEQ ID NO: 187 is the amino acid sequence of the LAD domain ofSWISSPROT:A0A1Q8LJS1 from Pseudonocardia sp Ae717_Ps2.

SEQ ID NO: 188 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:B2ASY2 from Podospora anserina.

SEQ ID NO: 189 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:B6GZX8 from Penicillium chrysogenum.

SEQ ID NO: 190 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:C7ZQ22 from Nectria haematococca.

SEQ ID NO: 191 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:E9DSA6 from Metarhizium acridum.

SEQ ID NO: 192 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:E9F1Z9 from Metarhizium robertsii.

SEQ ID NO: 193 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:E9FC42 from Metarhizium robertsii.

SEQ ID NO: 194 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:F9F2K5 from Fusarium oxysporum.

SEQ ID NO: 195 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:F9GF09 from Fusarium oxysporum.

SEQ ID NO: 196 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:G2QV10 from Thielavia terrestris.

SEQ ID NO: 197 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:G2QV26 from Thielavia terrestris.

SEQ ID NO: 198 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:J5TH48 from Trichosporon asahii var. Asahii.

SEQ ID NO: 199 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:J4UH35 from Trichosporon asahii var. Asahii.

SEQ ID NO: 200 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:J9NQ28 from Fusarium oxysporum f. sp. Lycopersici.

SEQ ID NO: 201 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:J9NQW0 from Fusarium oxysporum f. sp. Lycopersici.

SEQ ID NO: 202 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:K1VMN5 from Trichosporon asahii var. Asahii.

SEQ ID NO: 203 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:K1WL46 from Trichosporon asahii var. Asahii.

SEQ ID NO: 204 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9Z045 from Fusarium oxysporum f. sp. Melonis.

SEQ ID NO: 205 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:XOA5V9 from Fusarium oxysporum f. sp. Melonis.

SEQ ID NO: 206 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:N1S551 from Fusarium oxysporum f. sp. Cubense.

SEQ ID NO: 207 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:N4UD22 from Fusarium oxysporum f. sp. Cubense.

SEQ ID NO: 208 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:N4UT47 from Fusarium oxysporum f. sp. Cubense.

SEQ ID NO: 209 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9KVWV4 from Fusarium oxysporum.

SEQ ID NO: 210 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:X0MM97 from Fusarium oxysporum f. sp. Vasinfectum.

SEQ ID NO: 211 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W7N5Q6 from Gibberella moniliformis.

SEQ ID NO: 212 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:X0BE07 from Fusarium oxysporum f. sp. Raphani.

SEQ ID NO: 213 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:X0B118 from Fusarium oxysporum.

SEQ ID NO: 214 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9NW59 from Fusarium oxysporum f. sp. Pisi.

SEQ ID NO: 215 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9NXR4 from Fusarium oxysporum f. sp. Pisi.

SEQ ID NO: 216 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:S7ZNE7 from Penicillium oxalicum.

SEQ ID NO: 217 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:S7Z5Z6 from Penicillium oxalicum.

SEQ ID NO: 218 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9LDD0 from Fusarium oxysporum f. sp. Lycopersici.

SEQ ID NO: 219 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9HEM8 from Fusarium oxysporum.

SEQ ID NO: 220 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9JDH4 from Fusarium oxysporum.

SEQ ID NO: 221 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:S0EPI6 from Gibberella fujikuroi.

SEQ ID NO: 222 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W6QNL2 from Penicillium roqueforti.

SEQ ID NO: 223 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT: U4LJD9 from Pyronema omphalodes.

SEQ ID NO: 224 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:U4LG64 from Pyronema omphalodes.

SEQ ID NO: 225 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094AK50 from Pseudogymnoascus pannorum.

SEQ ID NO: 226 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9JIH2 from Fusarium oxysporum.

SEQ ID NO: 227 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:X0FW82 from Fusarium oxysporum f. sp.radicis-lycopersici.

SEQ ID NO: 228 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A086TBY7 from Acremonium chrysogenum.

SEQ ID NO: 229 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A086T755 from Acremonium chrysogenum.

SEQ ID NO: 230 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A086T4C8 from Acremonium chrysogenum.

SEQ ID NO: 231 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A086NNR4 from Metarhizium anisopliae.

SEQ ID NO: 232 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A086NFK5 from Metarhizium anisopliae.

SEQ ID NO: 233 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094FY19 from Pseudogymnoascus pannorum.

SEQ ID NO: 234 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094G1N0 from Pseudogymnoascus pannorum.

SEQ ID NO: 235 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094GEA0 from Pseudogymnoascus pannorum.

SEQ ID NO: 236 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094GJR5 from Pseudogymnoascus pannorum.

SEQ ID NO: 237 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094G660 from Pseudogymnoascus pannorum.

SEQ ID NO: 238 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A093YBN4 from Pseudogymnoascus pannorum.

SEQ ID NO: 239 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094FSZ5 from Pseudogymnoascus pannorum.

SEQ ID NO: 240 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094FBW1 from Pseudogymnoascus pannorum.

SEQ ID NO: 241 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094H0G2 from Pseudogymnoascus pannorum.

SEQ ID NO: 242 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094H7M1 from Pseudogymnoascus pannorum.

SEQ ID NO: 243 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A093Y8W3 from Pseudogymnoascus pannorum.

SEQ ID NO: 244 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094GYN9 from Pseudogymnoascus pannorum.

SEQ ID NO: 245 is the amino acid sequence of the lysozyme enhancingdomain of SWISS PROT:A0A093XAD4 from Pseudogymnoascus pannorum.

SEQ ID NO: 246 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094H7J6 from Pseudogymnoascus pannorum.

SEQ ID NO: 247 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094E0I1 from Pseudogymnoascus pannorum.

SEQ ID NO: 248 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094CC50 from Pseudogymnoascus pannorum.

SEQ ID NO: 249 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094II95 from Pseudogymnoascus pannorum.

SEQ ID NO: 250 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094IAA0 from Pseudogymnoascus pannorum.

SEQ ID NO: 251 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094IBC0 from Pseudogymnoascus pannorum.

SEQ ID NO: 252 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094E946 from Pseudogymnoascus pannorum.

SEQ ID NO: 253 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094A3A0 from Pseudogymnoascus pannorum.

SEQ ID NO: 254 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A1C4L9 from Aspergillus clavatus.

SEQ ID NO: 255 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A1CBV9 from Aspergillus clavatus.

SEQ ID NO: 256 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A1DA80 from Neosartorya fischeri.

SEQ ID NO: 257 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A1DBW2 from Neosartorya fischeri.

SEQ ID NO: 258 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A1DDF2 from Neosartorya fischeri.

SEQ ID NO: 259 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:Q0CED1 from Aspergillus terreus.

SEQ ID NO: 260 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:Q0CED2 from Aspergillus terreus.

SEQ ID NO: 261 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:Q0CV85 from Aspergillus terreus.

SEQ ID NO: 262 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:Q2GND8 from Chaetomium globosum.

SEQ ID NO: 263 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:Q2GND9 from Chaetomium globosum.

SEQ ID NO: 264 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:Q2H6W7 from Chaetomium globosum.

SEQ ID NO: 265 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:Q4WAY2 from Neosartorya fumigata.

SEQ ID NO: 266 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:Q4WBR4 from Neosartorya fumigata.

SEQ ID NO: 267 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:Q4WVY3 from Neosartorya fumigata.

SEQ ID NO: 268 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:B6H9X5 from Penicillium chrysogenum.

SEQ ID NO: 269 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:B6HR38 from Penicillium chrysogenum.

SEQ ID NO: 270 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:C7Z8W0 from Nectria haematococca.

SEQ ID NO: 271 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:C7ZQ20 from Nectria haematococca.

SEQ ID NO: 272 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:GORP87 from Hypocrea jecorina.

SEQ ID NO: 273 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:G2RG69 from Thielavia terrestris.

SEQ ID NO: 274 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:G2QNE9 from Thielavia heterothallica.

SEQ ID NO: 275 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:GORM22 from Hypocrea jecorina.

SEQ ID NO: 276 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:G0SG36 from Chaetomium thermophilum var.Thermophilum.

SEQ ID NO: 277 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:GORZV3 from Chaetomium thermophilum var.Thermophilum.

SEQ ID NO: 278 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:J9NQV9 from Fusarium oxysporum f. sp. Lycopersici.

SEQ ID NO: 279 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:G2QD02 from Thielavia heterothallica.

SEQ ID NO: 280 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:G2QNF0 from Thielavia heterothallica.

SEQ ID NO: 281 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:G9MHR1 from Hypocrea virens.

SEQ ID NO: 282 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:E9ELX9 from Metarhizium robertsii.

SEQ ID NO: 283 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:F9F2K4 from Fusarium oxysporum.

SEQ ID NO: 284 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:GORZV2 from Chaetomium thermophilum var.Thermophilum.

SEQ ID NO: 285 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:G2RG70 from Thielavia terrestris.

SEQ ID NO: 286 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9Z992 from Fusarium oxysporum f. sp. Melonis.

SEQ ID NO: 287 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9ZZW9 from Fusarium oxysporum f. sp. Melonis.

SEQ ID NO: 288 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9ZAE8 from Fusarium oxysporum f. sp. Melonis.

SEQ ID NO: 289 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:N1RWA4 from Fusarium oxysporum f. sp. Cubense.

SEQ ID NO: 290 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:N4UKT7 from Fusarium oxysporum f. sp. Cubense.

SEQ ID NO: 291 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W9KX02 from Fusarium oxysporum.

SEQ ID NO: 292 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:XOB4J3 from Fusarium oxysporum f. sp. Raphani.

SEQ ID NO: 293 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W6QE02 from Penicillium roqueforti.

SEQ ID NO: 294 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:W6R4X8 from Penicillium roqueforti.

SEQ ID NO: 295 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A024S9B8 from Trichoderma reesei.

SEQ ID NO: 296 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A086NN36 from Metarhizium anisopliae.

SEQ ID NO: 297 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094GA03 from Pseudogymnoascus pannorum.

SEQ ID NO: 298 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094C8U1 from Pseudogymnoascus pannorum.

SEQ ID NO: 299 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A093Z6Z8 from Pseudogymnoascus pannorum.

SEQ ID NO: 300 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094IML3 from Pseudogymnoascus pannorum.

SEQ ID NO: 301 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094GY79 from Pseudogymnoascus pannorum.

SEQ ID NO: 302 is the amino acid sequence of the lysozyme enhancingdomain of SWISS PROT:A0A093XPZ7 from Pseudogymnoascus pannorum.

SEQ ID NO: 303 is the amino acid sequence of the lysozyme enhancingdomain of SWISS PROT:A0A093XAS9 from Pseudogymnoascus pannorum.

SEQ ID NO: 304 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A09418J6 from Pseudogymnoascus pannorum.

SEQ ID NO: 305 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094FTL0 from Pseudogymnoascus pannorum.

SEQ ID NO: 306 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094AT39 from Pseudogymnoascus pannorum.

SEQ ID NO: 307 is the amino acid sequence of the lysozyme enhancingdomain of SWISS PROT:A0A093XSP5 from Pseudogymnoascus pannorum.

SEQ ID NO: 308 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094BAE6 from Pseudogymnoascus pannorum.

SEQ ID NO: 309 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A0941E25 from Pseudogymnoascus pannorum.

SEQ ID NO: 310 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094HNM8 from Pseudogymnoascus pannorum.

SEQ ID NO: 311 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094ETJ5 from Pseudogymnoascus pannorum.

SEQ ID NO: 312 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094EPJ7 from Pseudogymnoascus pannorum.

SEQ ID NO: 313 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094E9W0 from Pseudogymnoascus pannorum.

SEQ ID NO: 314 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094BWD6 from Pseudogymnoascus pannorum.

SEQ ID NO: 315 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A094BTS1 from Pseudogymnoascus pannorum.

SEQ ID NO: 316 is the amino acid sequence of the lysozyme enhancingdomain of SWISSPROT:A0A093ZTZ8 from Pseudogymnoascus pannorum.

SEQ ID NO: 317 is conserved motif I AG[I/L]AT[A/G][I/L][T/V]ES.

SEQ ID NO: 318 is conserved motif II V[G/A]XLCQXVQXSAYP.

SEQ ID NO: 319 is conserved motif III[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN].

SEQ ID NO: 320 is the synthetic DNA construct of plasmid pDAu770.

SEQ ID NO: 321 is the forward primer KKSC0972-F.

SEQ ID NO: 322 is the reverse primer KKSC0972-R.

SEQ ID NO: 323 is forward primer F1.

SEQ ID NO: 324 is reverse primer F1.

SEQ ID NO: 325 is forward primer F3.

SEQ ID NO: 326 is reverse primer F3.

SEQ ID NO: 327 Primer bind forward.

SEQ ID NO: 328 Primer bind reverse.

SEQ ID NO: 329 is the amino acid sequence of the truncated LYApolypeptide from Ovatospora brasiliensis.

FIGURES

FIG. 1 represents the map of the different DNA features included on theplasmid pDAu770. The amy2 locus flanking regions (3′ and 5′) areindicated by white boxes. Promoter regions are indicated by green boxesfor the promoter region of the pyrG, tef1 and tpi gene. The purple boxesindicate the selection cassette (ampR for ampicillin resistance and amdSfor acetamide selection). The terminator regions are indicated by blueboxes for the terminator region of the niaD and amg genes. The codingregion of the FLPase (sFLP) and the first exon of the pyrG gene areindicated in orange. The 5′ region of the pyrG intron is indicated ingrey. The origin of replication of the plasmid is indicated by ORI.

FIG. 2 is the schematic representation of transformation of the hoststrain DAu785 by the transforming DNA (either plasmid pDAu724 orderivatives or OverlapExtension PCR products.

Top panel represents the locus amy2 with the integration of the FLPlanding pad composed of FRT-F and FRT-F3 the FLPase recognition site, aswell as the amdS (acetamide) selection marker and the FLPase expressioncassette. A split PyrG marker has been used and at the amy2 locus the 5′end of the pyrG marker is inserted.

Middle panel represents the transforming DNA, in particular the regionthat is integrated at the FLP landing pad by site specific recombinationmediated by FLPase. The palsmid or PCR product must contain FRT-F and F3sites as well as the remaining 3′ part of the pyrG marker.

Bottom panel represents the resulting amy2 locus after site specificintegration of the transformaing DNA between the FRT sites. The amdS andFLP cassettes have been exchanged with the GOI expression cassette andthe 3′ part of the pyrG marker reconstituting a fully functionalselection marker.

Definitions

Animal: The term “animal” refers to any animal except humans. Examplesof animals are monogastric animals, including but not limited to pigs orswine (including, but not limited to, piglets, growing pigs, and sows);poultry such as turkeys, ducks, quail, guinea fowl, geese, pigeons(including squabs) and chicken (including but not limited to broilerchickens (referred to herein as broiles), chicks, layer hens (referredto herein as layers)); horses (including but not limited to hotbloods,coldbloods and warm bloods) crustaceans (including but not limited toshrimps and prawns) and fish (including but not limited to amberjack,arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama,carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie,dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut,java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet,paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa,sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook,sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia,trout, tuna, turbot, vendace, walleye and whitefish).

Animal feed: The term “animal feed” refers to any compound, preparation,or mixture suitable for, or intended for intake by a monogastric animal.Animal feed for a monogastric animal typically comprises concentrates aswell as vitamins, minerals, enzymes, direct fed microbial, amino acidsand/or other feed ingredients (such as in a premix).

Antimicrobial activity: The term “antimicrobial activity” is definedherein as an activity that kills or inhibits the growth ofmicroorganisms, such as, algae, archea, bacteria, fungi and/orprotozoans. The antimicrobial activity can, for example, be bactericidalmeaning the killing of bacteria or bacteriostatic meaning the preventionof bacterial growth. The antimicrobial activity can include catalyzingthe hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid andN-acetyl-D-glucosamine residues in a peptidoglycan and betweenN-acetyl-D-glucosamine residues in chitodextrins. Antimicrobial activitycan also include the LYS polypeptide binding to the surface of themicroorganism and inhibiting its growth. The antimicrobial effect canalso include the use of the LYS polypeptides of the present inventionfor activation of bacterial autolysins, as an immunostimulator, byinhibiting or reducing bacterial toxins and by an opsonin effect.

Body Weight Gain: The term “body weight gain” means an increase in liveweight of an animal during a given period of time e.g. the increase inweight from day 1 to day 21.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Concentrates: The term “concentrates” means feed with high protein andenergy concentrations, such as fish meal, molasses, oligosaccharides,sorghum, seeds and grains (either whole or prepared by crushing,milling, etc. from e.g. corn, oats, rye, barley, wheat), oilseed presscake (e.g. from cottonseed, safflower, sunflower, soybean (such assoybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake,yeast derived material and distillers grains (such as wet distillersgrains (WDS) and dried distillers grains with solubles (DDGS)).

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

European Production Efficacy Factor (EPEF): The “European ProductionEfficacy Factor” is a way of comparing the performance of animals. Thissingle-figure facilitates comparison of performance within and amongfarms and can be used to assess environmental, climatic and managementalvariables. The EPEF is calculated as [(liveability (%)×Liveweight(kg))/(Age at depletion (days)×FCR)]×100, wherein livability is thepercentage of animals alive at slaughter, Liveweight is the averageweight of the animals at slaughter, age of depletion is the age of theanimals at slaughter and FCR is the feed conversion ratio at slaughter.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Feed Conversion Ratio (FCR): FCR is a measure of an animal's efficiencyin converting feed mass into increases of the desired output. Animalsraised for meat—such as swine, poultry and fish—the output is the massgained by the animal. Specifically, FCR is calculated as feed intakedivided by weight gain, all over a specified period. Improvement in FCRmeans reduction of the FCR value. A FCR improvement of 2% means that theFCR was reduced by 2%.

Feed efficiency: The term “feed efficiency” means the amount of weightgain per unit of feed when the animal is fed ad-libitum or a specifiedamount of food during a period of time. By “increased feed efficiency”it is meant that the use of a feed additive composition according thepresent invention in feed results in an increased weight gain per unitof feed intake compared with an animal fed without said feed additivecomposition being present.

Forage: The term “forage” as defined herein also includes roughage.Forage is fresh plant material such as hay and silage from forageplants, grass and other forage plants, seaweed, sprouted grains andlegumes, or any combination thereof. Examples of forage plants areAlfalfa (lucerne), birdsfoot trefoil, brassica (e.g. kale, rapeseed(canola), rutabaga (swede), turnip), clover (e.g. alsike clover, redclover, subterranean clover, white clover), grass (e.g. Bermuda grass,brome, false oat grass, fescue, heath grass, meadow grasses, orchardgrass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats,rye, sorghum, soybeans and wheat and vegetables such as beets. Foragefurther includes crop residues from grain production (such as cornstover; straw from wheat, barley, oat, rye and other grains); residuesfrom vegetables like beet tops; residues from oilseed production likestems and leaves form soy beans, rapeseed and other legumes; andfractions from the refining of grains for animal or human consumption orfrom fuel production or other industries.

Fragment: The term “fragment” means a LYS polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide or domain; wherein the fragment haslysozyme activity.

In one aspect, the fragment comprises at least 90% of the length of themature polypeptide, such as at least 203 amino acids of SEQ ID NO: 2, atleast 203 amino acids of SEQ ID NO: 3, at least 203 amino acids of SEQID NO: 5, at least 203 amino acids of SEQ ID NO: 6, at least 200 aminoacids of SEQ ID NO: 8, at least 200 amino acids of SEQ ID NO: 9, atleast 273 amino acids of SEQ ID NO: 11, at least 273 amino acids of SEQID NO: 12, at least 205 amino acids of SEQ ID NO: 14, at least 205 aminoacids of SEQ ID NO: 15, at least 207 amino acids of SEQ ID NO: 17, atleast 207 amino acids of SEQ ID NO: 18, at least 207 amino acids of SEQID NO: 20, at least 207 amino acids of SEQ ID NO: 21, at least 208 aminoacids of SEQ ID NO: 23, at least 208 amino acids of SEQ ID NO: 24, atleast 205 amino acids of SEQ ID NO: 26, at least 205 amino acids of SEQID NO: 27, at least 205 amino acids of SEQ ID NO: 29, at least 205 aminoacids of SEQ ID NO: 30, at least 203 amino acids of SEQ ID NO: 32, atleast 203 amino acids of SEQ ID NO: 33, at least 202 amino acids of SEQID NO: 35, at least 202 amino acids of SEQ ID NO: 36, at least 202 aminoacids of SEQ ID NO: 38, at least 202 amino acids of SEQ ID NO: 39, atleast 273 amino acids of SEQ ID NO: 41, at least 273 amino acids of SEQID NO: 42, at least 204 amino acids of SEQ ID NO: 44, or at least 204amino acids of SEQ ID NO: 45.

In one aspect, the fragment comprises at least 92% of the length of themature polypeptide, such as at least 207 amino acids of SEQ ID NO: 2, atleast 207 amino acids of SEQ ID NO: 3, at least 207 amino acids of SEQID NO: 5, at least 207 amino acids of SEQ ID NO: 6, at least 205 aminoacids of SEQ ID NO: 8, at least 205 amino acids of SEQ ID NO: 9, atleast 279 amino acids of SEQ ID NO: 11, at least 279 amino acids of SEQID NO: 12, at least 209 amino acids of SEQ ID NO: 14, at least 209 aminoacids of SEQ ID NO: 15, at least 211 amino acids of SEQ ID NO: 17, atleast 211 amino acids of SEQ ID NO: 18, at least 211 amino acids of SEQID NO: 20, at least 211 amino acids of SEQ ID NO: 21, at least 213 aminoacids of SEQ ID NO: 23, at least 213 amino acids of SEQ ID NO: 24, atleast 209 amino acids of SEQ ID NO: 26, at least 209 amino acids of SEQID NO: 27, at least 209 amino acids of SEQ ID NO: 29, at least 209 aminoacids of SEQ ID NO: 30, at least 207 amino acids of SEQ ID NO: 32, atleast 207 amino acids of SEQ ID NO: 33, at least 207 amino acids of SEQID NO: 35, at least 207 amino acids of SEQ ID NO: 36, at least 207 aminoacids of SEQ ID NO: 38, at least 207 amino acids of SEQ ID NO: 39, atleast 279 amino acids of SEQ ID NO: 41, at least 279 amino acids of SEQID NO: 42, at least 208 amino acids of SEQ ID NO: 44, or at least 208amino acids of SEQ ID NO: 45.

In one aspect, the fragment comprises at least 94% of the length of themature polypeptide, such as at least 212 amino acids of SEQ ID NO: 2, atleast 212 amino acids of SEQ ID NO: 3, at least 212 amino acids of SEQID NO: 5, at least 212 amino acids of SEQ ID NO: 6, at least 209 aminoacids of SEQ ID NO: 8, at least 209 amino acids of SEQ ID NO: 9, atleast 285 amino acids of SEQ ID NO: 11, at least 285 amino acids of SEQID NO: 12, at least 214 amino acids of SEQ ID NO: 14, at least 214 aminoacids of SEQ ID NO: 15, at least 216 amino acids of SEQ ID NO: 17, atleast 216 amino acids of SEQ ID NO: 18, at least 216 amino acids of SEQID NO: 20, at least 216 amino acids of SEQ ID NO: 21, at least 218 aminoacids of SEQ ID NO: 23, at least 218 amino acids of SEQ ID NO: 24, atleast 214 amino acids of SEQ ID NO: 26, at least 214 amino acids of SEQID NO: 27, at least 214 amino acids of SEQ ID NO: 29, at least 214 aminoacids of SEQ ID NO: 30, at least 212 amino acids of SEQ ID NO: 32, atleast 212 amino acids of SEQ ID NO: 33, at least 211 amino acids of SEQID NO: 35, at least 211 amino acids of SEQ ID NO: 36, at least 211 aminoacids of SEQ ID NO: 38, at least 211 amino acids of SEQ ID NO: 39, atleast 285 amino acids of SEQ ID NO: 41, at least 285 amino acids of SEQID NO: 42, at least 213 amino acids of SEQ ID NO: 44, or at least 213amino acids of SEQ ID NO: 45.

In one aspect, the fragment comprises at least 96% of the length of themature polypeptide, such as at least 216 amino acids of SEQ ID NO: 2, atleast 216 amino acids of SEQ ID NO: 3, at least 216 amino acids of SEQID NO: 5, at least 216 amino acids of SEQ ID NO: 6, at least 214 aminoacids of SEQ ID NO: 8, at least 214 amino acids of SEQ ID NO: 9, atleast 291 amino acids of SEQ ID NO: 11, at least 291 amino acids of SEQID NO: 12, at least 218 amino acids of SEQ ID NO: 14, at least 218 aminoacids of SEQ ID NO: 15, at least 220 amino acids of SEQ ID NO: 17, atleast 220 amino acids of SEQ ID NO: 18, at least 220 amino acids of SEQID NO: 20, at least 220 amino acids of SEQ ID NO: 21, at least 222 aminoacids of SEQ ID NO: 23, at least 222 amino acids of SEQ ID NO: 24, atleast 218 amino acids of SEQ ID NO: 26, at least 218 amino acids of SEQID NO: 27, at least 218 amino acids of SEQ ID NO: 29, at least 218 aminoacids of SEQ ID NO: 30, at least 216 amino acids of SEQ ID NO: 32, atleast 216 amino acids of SEQ ID NO: 33, at least 216 amino acids of SEQID NO: 35, at least 216 amino acids of SEQ ID NO: 36, at least 216 aminoacids of SEQ ID NO: 38, at least 216 amino acids of SEQ ID NO: 39, atleast 291 amino acids of SEQ ID NO: 41, at least 291 amino acids of SEQID NO: 42, at least 217 amino acids of SEQ ID NO: 44, or at least 217amino acids of SEQ ID NO: 45.

In one aspect, the fragment comprises at least 98% of the length of themature polypeptide, such as at least 221 amino acids of SEQ ID NO: 2, atleast 221 amino acids of SEQ ID NO: 3, at least 221 amino acids of SEQID NO: 5, at least 221 amino acids of SEQ ID NO: 6, at least 218 aminoacids of SEQ ID NO: 8, at least 218 amino acids of SEQ ID NO: 9, atleast 297 amino acids of SEQ ID NO: 11, at least 297 amino acids of SEQID NO: 12, at least 223 amino acids of SEQ ID NO: 14, at least 223 aminoacids of SEQ ID NO: 15, at least 225 amino acids of SEQ ID NO: 17, atleast 225 amino acids of SEQ ID NO: 18, at least 225 amino acids of SEQID NO: 20, at least 225 amino acids of SEQ ID NO: 21, at least 227 aminoacids of SEQ ID NO: 23, at least 227 amino acids of SEQ ID NO: 24, atleast 223 amino acids of SEQ ID NO: 26, at least 223 amino acids of SEQID NO: 27, at least 223 amino acids of SEQ ID NO: 29, at least 223 aminoacids of SEQ ID NO: 30, at least 221 amino acids of SEQ ID NO: 32, atleast 221 amino acids of SEQ ID NO: 33, at least 220 amino acids of SEQID NO: 35, at least 220 amino acids of SEQ ID NO: 36, at least 220 aminoacids of SEQ ID NO: 38, at least 220 amino acids of SEQ ID NO: 39, atleast 297 amino acids of SEQ ID NO: 41, at least 297 amino acids of SEQID NO: 42, at least 222 amino acids of SEQ ID NO: 44, or at least 222amino acids of SEQ ID NO: 45.

In one aspect, the fragment comprises at least 99% of the length of themature polypeptide, such as at least 223 amino acids of SEQ ID NO: 2, atleast 223 amino acids of SEQ ID NO: 3, at least 223 amino acids of SEQID NO: 5, at least 223 amino acids of SEQ ID NO: 6, at least 220 aminoacids of SEQ ID NO: 8, at least 220 amino acids of SEQ ID NO: 9, atleast 300 amino acids of SEQ ID NO: 11, at least 300 amino acids of SEQID NO: 12, at least 225 amino acids of SEQ ID NO: 14, at least 225 aminoacids of SEQ ID NO: 15, at least 227 amino acids of SEQ ID NO: 17, atleast 227 amino acids of SEQ ID NO: 18, at least 227 amino acids of SEQID NO: 20, at least 227 amino acids of SEQ ID NO: 21, at least 229 aminoacids of SEQ ID NO: 23, at least 229 amino acids of SEQ ID NO: 24, atleast 225 amino acids of SEQ ID NO: 26, at least 225 amino acids of SEQID NO: 27, at least 225 amino acids of SEQ ID NO: 29, at least 225 aminoacids of SEQ ID NO: 30, at least 223 amino acids of SEQ ID NO: 32, atleast 223 amino acids of SEQ ID NO: 33, at least 222 amino acids of SEQID NO: 35, at least 222 amino acids of SEQ ID NO: 36, at least 222 aminoacids of SEQ ID NO: 38, at least 222 amino acids of SEQ ID NO: 39, atleast 300 amino acids of SEQ ID NO: 41, at least 300 amino acids of SEQID NO: 42, at least 224 amino acids of SEQ ID NO: 44, or at least 224amino acids of SEQ ID NO: 45.

Fusion polypeptide: The term “fusion polypeptide” is a polypeptide inwhich one polypeptide is fused at the N-terminus or the C-terminus ofthe polypeptide of the present invention. A fusion polypeptide isproduced by fusing a polynucleotide encoding another polypeptide to apolynucleotide of the present invention. Techniques for producing fusionpolypeptides are known in the art, and include ligating the codingsequences encoding the polypeptides so that they are in frame and thatexpression of the fusion polypeptide is under control of the samepromoter(s) and terminator. Fusion polypeptides may also be constructedusing intein technology in which fusion polypeptides are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779). A fusion polypeptide can furthercomprise a cleavage site between the two polypeptides. Upon secretion ofthe fusion protein, the site is cleaved releasing the two polypeptides.Examples of cleavage sites include, but are not limited to, the sitesdisclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251;Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493;Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al.,1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carteret al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248;and Stevens, 2003, Drug Discovery World 4: 35-48.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Hybrid polypeptide: The term “hybrid polypeptide” means a polypeptidecomprising domains from two or more polypeptides, e.g., a binding domainfrom one polypeptide and a catalytic domain from another polypeptide.The domains may be fused at the N-terminus or the C-terminus.

Isolated: The term “isolated” means a substance in a form that does notoccur in nature or in an environment in which the substance does notoccur in nature. Non-limiting examples of isolated substances include(1) any non-naturally occurring substance, (2) any substance including,but not limited to, any enzyme, variant, nucleic acid, protein, peptideor cofactor, that is at least partially removed from one or more or allof the naturally occurring constituents with which it is associated innature; (3) any substance modified by the hand of man relative to thatsubstance found in nature; or (4) any substance modified by increasingthe amount of the substance relative to other components with which itis naturally associated (e.g., recombinant production in a host cell;multiple copies of a gene encoding the substance; and use of a strongerpromoter than the promoter naturally associated with the gene encodingthe substance).

Lysozyme activity: The term “lysozyme activity” means the hydrolysis ofthe 1,4-beta-linkages between N-acetylmuramic acid andN-acetyl-D-glucosamine residues in a peptidoglycan, resulting inbacteriolysis. Lysozyme belongs to the enzyme class EC 3.2.1.17.Lysozyme activity is typically measured by the lytic action of thelysozyme on Micrococcus luteus ATCC 4698. In appropriate experimentalconditions these changes are proportional to the amount of lysozyme inthe medium (c.f. INS 1105 of the Combined Compendium of Food AdditiveSpecifications of the Food and Agriculture Organisation of the UN(www.fao.org)). For the purpose of the present invention, lysozymeactivity is determined according to the reducing-ends assay described inExample 1 (“Determination of Lysozyme Activity using reducing endsassay”). The polypeptide has lysozyme activity if it shows activityagainst Micrococcus luteus ATCC 4698.

In one aspect, the polypeptides of the present invention have at least50%, e.g., preferably at least 60%, preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, even morepreferably at least 95% or most preferably at least 100% of the lysozymeactivity of SEQ ID NO: 12, preferably wherein lysozyme activity isdetermined as described in Example 1. In one aspect, the polypeptides ofthe present invention have at least 50%, e.g., preferably at least 60%,preferably at least 70%, more preferably at least 80%, more preferablyat least 90%, even more preferably at least 95% or most preferably atleast 100% of the lysozyme activity of SEQ ID NO: 12 wherein lysozymeactivity is determined as follows: LYS polypeptide (50 μL of 0.7 μg/mLLYS polypeptide in phosphate buffer (5 mM citrate, 5 mM K₂HPO₄, 0.01%TritonX-100, pH 5.0)) is mixed with Micrococcus lysodeikticus solution(450 μL of 1% lyophilized Micrococcus lysodeikticus ATCC No. 4698 inmilli-Q water) and incubated at 40° C. with shaking (500 rpm) for 45min; the sample is centrifuged (4000 g, 5 min); supernatant (100 μL) ismixed with HCl (50 μL 3.2M) and incubated at 95° C. for 80 min; NaOH (50μL, 3.5 M) is added and 150 μL of the sample is added to4-hydroxybenzhydrazide in K—Na tartrate/NaOH buffer (75 μL of 50 g/LK—Na tartrate+20 g/L NaOH); the mixture is incubated at 95° C. for 10min; and the optical density is measured at 405 nm.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc.

In one aspect, the mature polypeptide is amino acids 1 to 226 of SEQ IDNO: 2 and amino acids −19 to −1 of SEQ ID NO: 2 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 1 to 226 of SEQ IDNO: 3. In one aspect, the mature polypeptide is amino acids 1 to 226 ofSEQ ID NO: 5 and amino acids −19 to −1 of SEQ ID NO: 5 are a signalpeptide. In another aspect, the mature polypeptide is amino acids 1 to226 of SEQ ID NO: 6. In one aspect, the mature polypeptide is aminoacids 1 to 223 of SEQ ID NO: 8 and amino acids −20 to −1 of SEQ ID NO: 8are a signal peptide. In another aspect, the mature polypeptide is aminoacids 1 to 223 of SEQ ID NO: 9. In one aspect, the mature polypeptide isamino acids 1 to 304 of SEQ ID NO: 11 and amino acids −20 to −1 of SEQID NO: 11 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 1 to 304 of SEQ ID NO: 12. In one aspect, themature polypeptide is amino acids 1 to 228 of SEQ ID NO: 14 and aminoacids −19 to −1 of SEQ ID NO: 14 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 1 to 228 of SEQ ID NO: 15.In one aspect, the mature polypeptide is amino acids 1 to 230 of SEQ IDNO: 17 and amino acids −20 to −1 of SEQ ID NO: 17 are a signal peptide.In another aspect, the mature polypeptide is amino acids 1 to 230 of SEQID NO: 18. In one aspect, the mature polypeptide is amino acids 1 to 230of SEQ ID NO: 20 and amino acids −21 to −1 of SEQ ID NO: 20 are a signalpeptide. In another aspect, the mature polypeptide is amino acids 1 to230 of SEQ ID NO: 21. In one aspect, the mature polypeptide is aminoacids 1 to 232 of SEQ ID NO: 23 and amino acids −22 to −1 of SEQ ID NO:23 are a signal peptide. In another aspect, the mature polypeptide isamino acids 1 to 232 of SEQ ID NO: 24. In one aspect, the maturepolypeptide is amino acids 1 to 228 of SEQ ID NO: 26 and amino acids −20to −1 of SEQ ID NO: 26 are a signal peptide. In another aspect, themature polypeptide is amino acids 1 to 228 of SEQ ID NO: 27. In oneaspect, the mature polypeptide is amino acids 1 to 228 of SEQ ID NO: 29and amino acids −20 to −1 of SEQ ID NO: 29 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 1 to 228 of SEQ IDNO: 30. In one aspect, the mature polypeptide is amino acids 1 to 226 ofSEQ ID NO: 32 and amino acids −19 to −1 of SEQ ID NO: 32 are a signalpeptide. In another aspect, the mature polypeptide is amino acids 1 to226 of SEQ ID NO: 33. In one aspect, the mature polypeptide is aminoacids 1 to 225 of SEQ ID NO: 35 and amino acids −20 to −1 of SEQ ID NO:35 are a signal peptide. In another aspect, the mature polypeptide isamino acids 1 to 225 of SEQ ID NO: 36. In one aspect, the maturepolypeptide is amino acids 1 to 225 of SEQ ID NO: 38 and amino acids −19to −1 of SEQ ID NO: 38 are a signal peptide. In another aspect, themature polypeptide is amino acids 1 to 225 of SEQ ID NO: 39. In oneaspect, the mature polypeptide is amino acids 1 to 304 of SEQ ID NO: 41and amino acids −19 to −1 of SEQ ID NO: 41 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 1 to 304 of SEQ IDNO: 42. In one aspect, the mature polypeptide is amino acids 1 to 227 ofSEQ ID NO: 44 and amino acids −19 to −1 of SEQ ID NO: 44 are a signalpeptide. In another aspect, the mature polypeptide is amino acids 1 to227 of SEQ ID NO: 45.

It is known in the art that a host cell may produce a mixture of two ofmore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide. Itis also known in the art that different host cells process polypeptidesdifferently, and thus, one host cell expressing a polynucleotide mayproduce a different mature polypeptide (e.g., having a differentC-terminal and/or N-terminal amino acid) as compared to another hostcell expressing the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving lysozyme activity.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Obtained or obtainable from: The term “obtained or obtainable from”means that the polypeptide may be found in an organism from a specifictaxonomic rank. In one embodiment, the polypeptide is obtained orobtainable from the kingdom Fungi, wherein the term kingdom is thetaxonomic rank. In a preferred embodiment, the polypeptide is obtainedor obtainable from the phylum Ascomycota, wherein the term phylum is thetaxonomic rank. In another preferred embodiment, the polypeptide isobtained or obtainable from the subphylum Pezizomycotina, wherein theterm subphylum is the taxonomic rank.

If the taxonomic rank of a polypeptide is not known, it can easily bedetermined by a person skilled in the art by performing a BLASTP searchof the polypeptide (using e.g. the National Center for BiotechnologyInformation (NCIB) website http://www.ncbi.nlm.nih.gov/) and comparingit to the closest homologues. An unknown polypeptide which is a fragmentof a known polypeptide is considered to be of the same taxonomicspecies. An unknown natural polypeptide or artificial variant whichcomprises a substitution, deletion and/or insertion in up to 10positions is considered to be from the same taxonomic species as theknown polypeptide.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Roughage: The term “roughage” means dry plant material with high levelsof fiber, such as fiber, bran, husks from seeds and grains and cropresidues (such as stover, copra, straw, chaff, sugar beet waste).

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabelled “longest identity” (obtained using the—nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labelled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having lysozyme activity.

Substantially pure polypeptide: The term “substantially purepolypeptide” means a preparation that contains at most 10%, at most 8%,at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%,and at most 0.5% by weight of other polypeptide material with which itis natively or recombinantly associated. Preferably, the polypeptide isat least 92% pure, e.g., at least 94% pure, at least 95% pure, at least96% pure, at least 97% pure, at least 98% pure, at least 99%, at least99.5% pure, and 100% pure by weight of the total polypeptide materialpresent in the preparation. The polypeptides of the present inventionare preferably in a substantially pure form. This can be accomplished,for example, by preparing the polypeptide by well known recombinantmethods or by classical purification methods.

Variant: The term “variant” means a polypeptide having lysozyme activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, of one or more (several) amino acid residues at one or more(e.g., several) positions. A substitution means replacement of the aminoacid occupying a position with a different amino acid; a deletion meansremoval of the amino acid occupying a position; and an insertion meansadding 1, 2, or 3 amino acids adjacent to and immediately following theamino acid occupying the position.

In one aspect, the variant according to the invention may comprise from1 to 5; from 1 to 10; from 1 to 15; from 1 to 20; from 1 to 25; from 1to 30; from 1 to 35; from 1 to 40; from 1 to 45; or from 1-50, i.e. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 alterations.

In one aspect, the variant of the present invention has at least 50%,e.g., preferably at least 60%, preferably at least 70%, more preferablyat least 80%, more preferably at least 90%, even more preferably atleast 95% or most preferably at least 100% of the lysozyme activity ofSEQ ID NO: 12, preferably wherein lysozyme activity is determined asdescribed in Example 1.

In one aspect, the variant of the present invention has at least 50%,e.g., preferably at least 60%, preferably at least 70%, more preferablyat least 80%, more preferably at least 90%, even more preferably atleast 95% or most preferably at least 100% of the lysozyme activity ofSEQ ID NO: 12 wherein lysozyme activity is determined as follows: LYSpolypeptide (50 μL of 0.7 μg/mL LYS polypeptide in phosphate buffer (5mM citrate, 5 mM K₂HPO₄, 0.01% TritonX-100, pH 5.0)) is mixed withMicrococcus lysodeikticus solution (450 μL of 1% lyophilized Micrococcuslysodeikticus ATCC No. 4698 in milli-Q water) and incubated at 40° C.with shaking (500 rpm) for 45 min; the sample is centrifuged (4000 g, 5min); supernatant (100 μL) is mixed with HCl (50 μL 3.2M) and incubatedat 95° C. for 80 min; NaOH (50 μL, 3.5 M) is added and 150 μL of thesample is added to 4-hydroxybenzhydrazide in K—Na tartrate/NaOH buffer(75 μL of 50 g/L K—Na tartrate+20 g/L NaOH); the mixture is incubated at95° C. for 10 min; and the optical density is measured at 405 nm.

In one aspect, the variant according to the invention may comprise from1 to 5; from 1 to 10; from 1 to 15; from 1 to 20; from 1 to 25; from 1to 30; from 1 to 35; from 1 to 40; from 1 to 45; or from 1-50, i.e. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 alterations and has atleast 50%, e.g., preferably at least 60%, preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, even morepreferably at least 95% or most preferably at least 100% of the lysozymeactivity of SEQ ID NO: 12, preferably wherein lysozyme activity isdetermined as described in Example 1.

Nomenclature

For purposes of the present invention, the nomenclature [E/Q] means thatthe amino acid at this position may be a glutamic acid (Glu, E) or aglutamine (Gln, Q). Likewise the nomenclature [V/G/A/I] means that theamino acid at this position may be a valine (Val, V), glycine (Gly, G),alanine (Ala, A) or isoleucine (Ile, I), and so forth for othercombinations as described herein. Unless otherwise limited further, theamino acid X is defined such that it may be any of the 20 natural aminoacids.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered a completely novel class of polypeptideshaving lysozyme activity. Said polypeptides are structurally quitedifferent from known lysozymes. As shown in the sequence identity matrixbelow, the polypeptides of the present invention all have a sequenceidentity less than 45% to the prior art sequences disclosed inWO2013/076259, suggesting that these novel polypeptides may have adifferent folding pattern to known lysozymes.

GH class SEQ3 SEQ2 SEQ4 SEQ6 SEQ8 HEWL SEQ ID NO: 3 of Not 100 27 33.343.8 23.3 30.93 present invention defined SEQ ID NO: 2 of GH23 27 100 2733.3 32 21.3 WO2013/076259 SEQ ID NO: 4 of GH24 33.3 27 100 79 32.733.33 WO2013/076259 SEQ ID NO: 6 of GH25 43.8 33.3 79 100 34.1 44.78WO2013/076259 SEQ ID NO: 8 of GH25 23.3 32 32.7 34.1 100 28.97WO2013/076259 Hen Egg White GH22 30.9 21.3 33.3 44.8 29 100 (SwissprotP00698)

The polypeptides of the present invention demonstrate typical lysozymeactivity such as activity in the traditional OD drop assay againstMicrococcus lysodeikticus (see example 14) or a reducing ends assayusing Micrococcus lysodeikticus as substrate (see example 13).

The polypeptides of the invention having lysozyme activity are hereinnamed LYS polypeptides and comprise one or more LAD (Lysozyme ActiveDomain) catalytic domains and optionally one or more lysozyme enhancingdomains (LED).

Compositions Comprising Polypeptides Having Lysozyme Activity

In the first aspect, the invention relates to a composition comprisingat least 0.01 mg of LYS polypeptide per kilogram of composition, whereinthe polypeptide (a) has lysozyme activity and (b) comprises one or moreLAD catalytic domains; wherein the LAD catalytic domain gives a domTscore of at least 180 when queried using a Profile Hidden Markov Model(HMM) prepared using SEQ ID NOs: 46 to 187 and hmmbuild softwareprogram, and wherein the query is carried out using hmmscan softwareprogram by the Method of Determining the LAD Catalytic Domain by HMM.

In an embodiment, the polypeptide further comprises one or more lysozymeenhancing domains (LED). Thus, the invention further relates to acomposition comprising at least 0.01 mg of LYS polypeptide per kilogramof composition, wherein:

-   -   (a) the LYS polypeptide has lysozyme activity;    -   (b) the LYS polypeptide comprises one or more LAD catalytic        domains; wherein the LAD catalytic domain gives a domT score of        at least 180 when queried using a Profile Hidden Markov Model        (HMM) prepared using SEQ ID NOs: 46 to 187 and hmmbuild software        program, and wherein the query is carried out using hmmscan        software program by the Method of Determining the LAD Catalytic        Domain by HMM;    -   (c) the polypeptide comprises one or more LED domains, wherein        the LED gives a domT score of at least 100 when queried using a        Profile Hidden Markov Model prepared using SEQ ID NOs: 188 to        316 and hmmbuild software program, and wherein the query is        carried out using the hmmscan software program.

The theory behind Profile HMMs as described in Durbin et al. (Biologicalsequence analysis: probabilistic models of proteins and nucleic acids,Cambridge University Press, 1998) and Krogh et al. (1994 J. Mol. Biol.235:1501-1531), both incorporated herein by reference, ischaracterization of a set of proteins based on the probability of eachamino acid occurring at each position in the alignment of the proteinsof the set.

Specifically, profile HMMs are statistical models of multiple sequencealignments, or even of single sequences. They capture position-specificinformation about how conserved each column of the alignment is, andwhich residues are likely. All profile methods are more or lessstatistical descriptions of the consensus of a multiple sequencealignment. They use position-specific scores for amino acids ornucleotides (residues) and position specific penalties for opening andextending an insertion or deletion. Traditional pairwise alignment (forexample, BLAST, FASTA or the Smith/Waterman algorithm) usesposition-independent scoring parameters. This property of profilescaptures important information about the degree of conservation atvarious positions in the multiple alignment, and the varying degree towhich gaps and insertions are permitted.

The advantage of using HMMs is that HMMs have a formal probabilisticbasis. Probability theory is used to guide how all the scoringparameters should be set. One of the most important aspect is that HMMshave a consistent theory for setting position-specific gap and insertionscores. The methods are consistent and therefore highly automatable,allowing hundreds of profile HMMs to be applied to e.g. whole genomeanalysis. An example of a protein domain model database is Pfam(Sonnhammer et al., 1997, ‘A comprehensive database of protein familiesbased on seed alignments’, Proteins, 28:405-420; Finn et al., 2010, ‘ThePfam protein families database’, Nucl. Acids Res., 38:D211-D222), whichis a significant part of the Interpro protein domain annotation system.The construction and use of Pfam is tightly tied to the HMM ER softwarepackage (see https://en.wikipedia.org/wiki/HMMER).

The LAD catalytic domain is defined in the following manner. SEQ ID NOs:46 to 187, which are partial sequences of the Uniprot entries asexplained in the ‘overview of sequence listing’ section herein, arealigned using the software program MUSCLE v3.8.31 with the defaultsettings. Using this alignment, a hidden Markov model (HMM) is built forthe LAD catalytic domain. The HMM is constructed using the softwareprogram ‘hmmbuild’ from the package HMMER 3.0 (March 2010)(http://hmmer.org/) and the software is invoked using the defaultsettings.

A LAD catalytic domain is defined to match the above mentioned HMM usingthe software program ‘hmmscan’ from the package HMMER 3.0 (March 2010)(http://hmmer.org/) using the default settings if the domT score is atleast 170. In a preferred embodiment, the domT score is at least 175,preferably at least 180, more preferably at least 185, even morepreferably at least 190, even more preferably at least 195, or mostpreferably at least 200.

The HMM profile of the LAD catalytic domain as generated using SEQ IDNOs: 46 to 187 according to the procedure above is given in example 10.The HMM profile can be copied into a text file which is subsequentlyloaded into the software program ‘hmmscan’ so that other polypeptidescan be tested to see whether said polypeptide comprises one or more LADcatalytic domains.

The Lysozyme Enhancing Domain (LED) is defined in the following manner.SEQ ID NOs: 188 to 316, which are partial sequences of the Uniprotentries as explained in the ‘overview of sequence listing’ sectionherein, are aligned using the software program MUSCLE v3.8.31 with thedefault settings. Using this alignment, a hidden Markov model (HMM) isbuilt for the LED. The HMM is constructed using the software program‘hmmbuild’ from the package HMMER 3.0 (March 2010) (http://hmmer.org/)and the software is invoked using the default settings.

A LED is defined to match the above mentioned HMM using the softwareprogram ‘hmmscan’ from the package HMMER 3.0 (March 2010)(http://hmmer.org/) using the default settings if the domT score is atleast 100. In a preferred embodiment, the domT score is at least 103,preferably at least 106, more preferably at least 109, more preferablyat least 112, more preferably at least 115, more preferably at least118, even more preferably at least 121, or most preferably at least 124.

The HMM profile of the LED as generated using SEQ ID NOs: 188 to 316according to the procedure above is given in example 11. The HMM profilecan be copied into a text file which is subsequently loaded into thesoftware program ‘hmmscan’ so that other polypeptides can be tested tosee whether said polypeptide comprises one or more LED.

In an embodiment, the LAD catalytic domain gives a domT score of atleast 175 and the LED gives a domT score of at least 100. In anembodiment, the LAD catalytic domain gives a domT score of at least 180and the LED gives a domT score of at least 100. In an embodiment, theLAD catalytic domain gives a domT score of at least 185 and the LEDgives a domT score of at least 100. In an embodiment, the LAD catalyticdomain gives a domT score of at least 190 and the LED gives a domT scoreof at least 100. In an embodiment, the LAD catalytic domain gives a domTscore of at least 195 and the LED gives a domT score of at least 100. Inan embodiment, the LAD catalytic domain gives a domT score of at least200 and the LED gives a domT score of at least 100.

In an embodiment, the LAD catalytic domain gives a domT score of atleast 175 and the LED gives a domT score of at least 103. In anembodiment, the LAD catalytic domain gives a domT score of at least 180and the LED gives a domT score of at least 103. In an embodiment, theLAD catalytic domain gives a domT score of at least 185 and the LEDgives a domT score of at least 103. In an embodiment, the LAD catalyticdomain gives a domT score of at least 190 and the LED gives a domT scoreof at least 103. In an embodiment, the LAD catalytic domain gives a domTscore of at least 195 and the LED gives a domT score of at least 103. Inan embodiment, the LAD catalytic domain gives a domT score of at least200 and the LED gives a domT score of at least 103.

In an embodiment, the LAD catalytic domain gives a domT score of atleast 175 and the LED gives a domT score of at least 106. In anembodiment, the LAD catalytic domain gives a domT score of at least 180and the LED gives a domT score of at least 106. In an embodiment, theLAD catalytic domain gives a domT score of at least 185 and the LEDgives a domT score of at least 106. In an embodiment, the LAD catalyticdomain gives a domT score of at least 190 and the LED gives a domT scoreof at least 106. In an embodiment, the LAD catalytic domain gives a domTscore of at least 195 and the LED gives a domT score of at least 106. Inan embodiment, the LAD catalytic domain gives a domT score of at least200 and the LED gives a domT score of at least 106.

In an embodiment, the LAD catalytic domain gives a domT score of atleast 175 and the LED gives a domT score of at least 109. In anembodiment, the LAD catalytic domain gives a domT score of at least 180and the LED gives a domT score of at least 109. In an embodiment, theLAD catalytic domain gives a domT score of at least 185 and the LEDgives a domT score of at least 109. In an embodiment, the LAD catalyticdomain gives a domT score of at least 190 and the LED gives a domT scoreof at least 109. In an embodiment, the LAD catalytic domain gives a domTscore of at least 195 and the LED gives a domT score of at least 109. Inan embodiment, the LAD catalytic domain gives a domT score of at least200 and the LED gives a domT score of at least 109.

In an embodiment, the LAD catalytic domain gives a domT score of atleast 175 and the LED gives a domT score of at least 112. In anembodiment, the LAD catalytic domain gives a domT score of at least 180and the LED gives a domT score of at least 112. In an embodiment, theLAD catalytic domain gives a domT score of at least 185 and the LEDgives a domT score of at least 112. In an embodiment, the LAD catalyticdomain gives a domT score of at least 190 and the LED gives a domT scoreof at least 112. In an embodiment, the LAD catalytic domain gives a domTscore of at least 195 and the LED gives a domT score of at least 112. Inan embodiment, the LAD catalytic domain gives a domT score of at least200 and the LED gives a domT score of at least 112.

In an embodiment, the LAD catalytic domain gives a domT score of atleast 175 and the LED gives a domT score of at least 115. In anembodiment, the LAD catalytic domain gives a domT score of at least 180and the LED gives a domT score of at least 115. In an embodiment, theLAD catalytic domain gives a domT score of at least 185 and the LEDgives a domT score of at least 115. In an embodiment, the LAD catalyticdomain gives a domT score of at least 190 and the LED gives a domT scoreof at least 115. In an embodiment, the LAD catalytic domain gives a domTscore of at least 195 and the LED gives a domT score of at least 115. Inan embodiment, the LAD catalytic domain gives a domT score of at least200 and the LED gives a domT score of at least 115.

In an embodiment, the LAD catalytic domain gives a domT score of atleast 175 and the LED gives a domT score of at least 118. In anembodiment, the LAD catalytic domain gives a domT score of at least 180and the LED gives a domT score of at least 118. In an embodiment, theLAD catalytic domain gives a domT score of at least 185 and the LEDgives a domT score of at least 118. In an embodiment, the LAD catalyticdomain gives a domT score of at least 190 and the LED gives a domT scoreof at least 118. In an embodiment, the LAD catalytic domain gives a domTscore of at least 195 and the LED gives a domT score of at least 118. Inan embodiment, the LAD catalytic domain gives a domT score of at least200 and the LED gives a domT score of at least 118.

In an embodiment, the LAD catalytic domain comprises one or more motifI: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, theLAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP(SEQ ID NO: 318). In an embodiment, the LED comprises one or more motifIII: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). Inan embodiment, the LAD catalytic domain comprises one or more motif I:AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317) and the LED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or moremotif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:319). In an embodiment, the LAD catalytic domain comprises one or moremotif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or moremotif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises oneor more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ IDNO: 319).

In one embodiment of the first aspect, the invention relates to acomposition comprising one or more LYS polypeptides having lysozymeactivity, wherein the polypeptide is dosed at least 0.01 mg ofpolypeptide per kilogram of composition and is selected from the groupconsisting of:

-   -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 3;    -   (b) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 27;    -   (j) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 36;    -   (m) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 42;    -   (o) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 45;    -   (p) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID        NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO:        24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36,        SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45        positions;    -   (q) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o)        or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;    -   (r) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o)        or (p) and a N-terminal and/or C-terminal extension of up to 10        amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;        and    -   (s) a fragment of the polypeptide of (a), (b), (c), (d), (e),        (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having        lysozyme activity and having at least 90% of the length of the        mature polypeptide.

In one embodiment of the first aspect, the invention relates to acomposition comprising one or more LYS polypeptides having lysozymeactivity, wherein the LYS polypeptide is dosed at least 0.01 mg ofpolypeptide per kilogram of composition and comprises a LAD catalyticdomain that is selected from the group consisting of:

-   -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 3;    -   (b) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 81 to        220 of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 161        to 304 of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 88 to        230 of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 87 to        230 of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 90 to        232 of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 27;    -   (j) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 83 to        222 of SEQ ID NO: 36;    -   (m) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 82 to        225 of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 161        to 303 of SEQ ID NO: 42; and    -   (o) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        227 of SEQ ID NO: 45.

In one embodiment of the first aspect, the invention relates to acomposition comprising one or more LYS polypeptides having lysozymeactivity, wherein the LYS polypeptide is dosed at least 0.01 mg ofpolypeptide per kilogram of composition and comprises a LAD catalyticdomain that is selected from the group consisting of:

-   -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 3;    -   (b) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 81 to        220 of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 161        to 304 of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 88 to        230 of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 87 to        230 of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 90 to        232 of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 27;    -   (j) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 83 to        222 of SEQ ID NO: 36;    -   (m) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 82 to        225 of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 161        to 303 of SEQ ID NO: 42; and    -   (o) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        227 of SEQ ID NO: 45;

and wherein the LYS polypeptide comprises a LED domain that is selectedfrom the group consisting of:

-   -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 3;    -   (b) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        72 of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 27;    -   (j) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 36;    -   (m) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        72 of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 42;    -   (o) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 45;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 96 to        167 of SEQ ID NO: 12; and    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 96 to        168 of SEQ ID NO: 42.

In one embodiment to any part of the first aspect, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317). In an embodiment, the LAD catalytic domain comprises one ormore motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, theLED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif I:AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317) and the LED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or moremotif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:319). In an embodiment, the LAD catalytic domain comprises one or moremotif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or moremotif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises oneor more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ IDNO: 319).

In one embodiment to any part of the first aspect, the polypeptide is offungal origin. In an embodiment, the polypeptide is obtained orobtainable from the taxonomic phylum Ascomycota, preferably thetaxonomic subphylum Pezizomycotina.

In one embodiment to any part of the first aspect, the compositioncomprises at least 0.01 mg of polypeptide (enzyme protein) per kilogramof composition, such as at least 0.02 mg, 0.05 mg, 0.10 mg, 0.2 mg, 0.5mg, 1.0 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1.0g, 2.5 g, 5 g, 7.5 g, 10 g, 25 g, 50 g, 75 g or 100 g per kilogram ofcomposition. In one embodiment, the composition comprises at most 250 gof polypeptide per kilogram of composition, such as at most 150 g, 100g, 50 g, 40 g, 30 g, 20 g, 10 g, 7.5 g, 5 g, 2.5 g, 1.0 g, 750 mg, 500mg, 250 mg, 100 mg, 50 mg, 25 mg, 10 mg, 5 mg, 2.5 mg or 1 mg perkilogram of composition. In one embodiment, the composition comprisesbetween 0.01 mg and 250 g of polypeptide (enzyme protein) per kilogramof composition, such as between 0.02 mg, 0.05 mg, 0.10 mg, 0.2 mg, 0.5mg, 1.0 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1.0g, 2.5 g, 5 g, 7.5 g, 10 g, 25 g, 50 g, 75 g or 100 g per kilogram ofcomposition and 150 g, 100 g, 50 g, 40 g, 30 g, 20 g, 10 g, 7.5 g, 5 g,2.5 g, 1.0 g, 750 mg, 500 mg, 250 mg, 100 mg, 50 mg, 25 mg, 10 mg, 5 mg,2.5 mg or 1 mg per kilogram of composition, or any combination thereof.

In one embodiment to any part of the first aspect, the compositioncomprises one or more formulating agents (such as those describedherein), preferably a formulating agent selected from the listconsisting of glycerol, ethylene glycol, 1, 2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate,sodium sulfate, potassium sulfate, magnesium sulfate, sodiumthiosulfate, calcium carbonate, sodium citrate, dextrin, glucose,sucrose, sorbitol, lactose, starch, kaolin, maltodextrin, cyclodextrin,wheat, PVA, acetate, phosphate and cellulose, preferably selected fromthe list consisting of 1, 2-propylene glycol, 1, 3-propylene glycol,sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin andcalcium carbonate.

In one embodiment to any part of the first aspect, the compositioncomprises one or more additional enzymes. The one or more additionalenzymes is preferably selected from the group consisting of acetyl xylanesterase, alpha-amylase, beta-amylase, arabinofuranosidase,cellobiohydrolases, cellulase, feruloyl esterase, galactanase,alpha-galactosidase, beta-galactosidase, beta-glucanase,beta-glucosidase, lipase, lysophospholipase, lysozyme, mannanase,alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1,phospholipase A2, phospholipase C, phospholipase D, protease,pullulanase, pectinase, pectin lyase, xylanase, beta-xylosidase or anycombination thereof.

In one embodiment to any part of the first aspect, the compositioncomprises one or more probiotics. The one or more probiotics ispreferably selected from the group consisting of Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus,Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacteriumanimalis, Bifidobacterium sp., Carnobacterium sp., Clostridiumbutyricum, Clostridium sp., Enterococcus faecium, Enterococcus sp.,Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus farciminus,Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillussalivarius, Lactococcus lactis, Lactococcus sp., Leuconostoc sp.,Megasphaera elsdenii, Megasphaera sp., Pediococcus acidilactici,Pediococcus sp., Propionibacterium thoenii, Propionibacterium sp. andStreptococcus sp. or any combination thereof.

Granules Comprising Polypeptides Having Lysozyme Activity

In a second aspect, the invention relates to a granule comprising a LYSpolypeptide, wherein the polypeptide (a) has lysozyme activity and (b)comprises one or more LAD catalytic domains; wherein the LAD catalyticdomain gives a domT score of at least 180 when queried using a ProfileHidden Markov Model (HMM) prepared using SEQ ID NOs: 46 to 187 andhmmbuild software program, and wherein the query is carried out usinghmmscan software program by the Method of Determining the LAD CatalyticDomain by HMM. In one embodiment, the granule comprises a core particleand one or more coatings. In a preferred embodiment, the coatingcomprises salt and/or wax and/or flour. Preferred formulations aredisclosed in the formulation section below.

In an embodiment, the polypeptide further comprises one or more lysozymeenhancing domains (LED). Thus, the invention further relates to agranule comprising a LYS polypeptide, wherein:

-   -   (a) the LYS polypeptide has lysozyme activity;    -   (b) the LYS polypeptide comprises one or more LAD catalytic        domains, wherein the LAD catalytic domain gives a domT score of        at least 170 when queried using a Profile Hidden Markov Model        prepared using SEQ ID NOs: 46 to 187 and hmmbuild software        program, and wherein the query is carried out using hmmscan        software program by the Method of Determining the LAD Catalytic        Domain by HMM;    -   (c) the polypeptide comprises one or more LED domains, wherein        the LED gives a domT score of at least 100 when queried using a        Profile Hidden Markov Model prepared using SEQ ID NOs: 188 to        316 and hmmbuild software program, and typically wherein the        query is carried out using the hmmscan software program by the        Method of Determining the Lysozyme Enhancing Domain by HMM.

In one embodiment, the granule comprises a core particle and one or morecoatings. In a preferred embodiment, the coating comprises salt and/orwax and/or flour. Preferred formulations are disclosed in theformulation section below.

In an embodiment, the domT score of the LAD catalytic domain is at least175, preferably at least 180, more preferably at least 185, even morepreferably at least 190, even more preferably at least 195, or mostpreferably at least 200. In an embodiment, the domT score of the LED isat least 103, preferably at least 106, more preferably at least 109,more preferably at least 112, more preferably at least 115, morepreferably at least 118, even more preferably at least 121, or mostpreferably at least 124. Preferred combinations of domT scores are asdisclosed in the first aspect of the invention.

In an embodiment, the LAD catalytic domain comprises one or more motifI: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, theLAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP(SEQ ID NO: 318). In an embodiment, the LED comprises one or more motifIII: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). Inan embodiment, the LAD catalytic domain comprises one or more motif I:AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317) and the LED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or moremotif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:319). In an embodiment, the LAD catalytic domain comprises one or moremotif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or moremotif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises oneor more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ IDNO: 319).

In one embodiment of the second aspect, the invention relates to agranule comprising one or more LYS polypeptides having lysozymeactivity, wherein the LYS polypeptide is selected from the groupconsisting of:

-   -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 3;    -   (b) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 27;    -   (j) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 36;    -   (m) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 42;    -   (o) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 45;    -   (p) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID        NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO:        24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36,        SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45        positions;    -   (q) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o)        or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;    -   (r) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o)        or (p) and a N-terminal and/or C-terminal extension of up to 10        amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;        and    -   (s) a fragment of the polypeptide of (a), (b), (c), (d), (e),        (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having        lysozyme activity and having at least 90% of the length of the        mature polypeptide.

In one embodiment, the granule comprises a core particle and one or morecoatings. In a preferred embodiment, the coating comprises salt and/orwax and/or flour. Preferred formulations are disclosed in theformulation section below.

In one embodiment of the second aspect, the invention relates to agranule comprising one or more LYS polypeptides having lysozymeactivity, wherein the LYS polypeptide comprises a LAD catalytic domainthat is selected from the group consisting of:

-   -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 3;    -   (b) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 81 to        220 of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 161        to 304 of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 88 to        230 of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 87 to        230 of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 90 to        232 of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 27;    -   (j) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 83 to        222 of SEQ ID NO: 36;    -   (m) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 82 to        225 of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 161        to 303 of SEQ ID NO: 42; and    -   (o) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        227 of SEQ ID NO: 45.

In one embodiment of the second aspect, the invention relates to agranule comprising one or more LYS polypeptides having lysozymeactivity, wherein the LYS polypeptide comprises a LAD catalytic domainthat is selected from the group consisting of:

-   -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 3;    -   (b) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 81 to        220 of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 161        to 304 of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 88 to        230 of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 87 to        230 of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 90 to        232 of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 27;    -   (j) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        228 of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 84 to        226 of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 83 to        222 of SEQ ID NO: 36;    -   (m) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 82 to        225 of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 161        to 303 of SEQ ID NO: 42; and    -   (o) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 85 to        227 of SEQ ID NO: 45;

and wherein the LYS polypeptide comprises a LED domain that is selectedfrom the group consisting of:

-   -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 3;    -   (b) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        72 of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 27;    -   (j) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 36;    -   (m) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        72 of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 42;    -   (o) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 1 to        73 of SEQ ID NO: 45;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 96 to        167 of SEQ ID NO: 12; and    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 90% or at least 95% sequence identity to amino acids 96 to        168 of SEQ ID NO: 42.

In one embodiment, the granule comprises a core particle and one or morecoatings. In a preferred embodiment, the coating comprises salt and/orwax and/or flour. Preferred formulations are disclosed in theformulation section below.

In one embodiment to any part of the second aspect, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317). In an embodiment, the LAD catalytic domain comprises one ormore motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, theLED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif I:AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317) and the LED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or moremotif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:319). In an embodiment, the LAD catalytic domain comprises one or moremotif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or moremotif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises oneor more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ IDNO: 319).

In one embodiment to any part of the second aspect, the polypeptide isof fungal origin. In an embodiment, the polypeptide is obtained orobtainable from the taxonomic phylum Ascomycota, preferably thetaxonomic subphylum Pezizomycotina.

In one embodiment to any part of the first aspect, the compositioncomprises at least 0.01 mg of polypeptide (enzyme protein) per kilogramof composition, such as at least 0.02 mg, 0.05 mg, 0.10 mg, 0.2 mg, 0.5mg, 1.0 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1.0g, 2.5 g, 5 g, 7.5 g, 10 g, 25 g, 50 g, 75 g or 100 g per kilogram ofcomposition. In one embodiment, the composition comprises at most 250 gof polypeptide per kilogram of composition, such as at most 150 g, 100g, 50 g, 40 g, 30 g, 20 g, 10 g, 7.5 g, 5 g, 2.5 g, 1.0 g, 750 mg, 500mg, 250 mg, 100 mg, 50 mg, 25 mg, 10 mg, 5 mg, 2.5 mg or 1 mg perkilogram of composition. In one embodiment, the composition comprisesbetween 0.01 mg and 250 g of polypeptide (enzyme protein) per kilogramof composition, such as between 0.02 mg, 0.05 mg, 0.10 mg, 0.2 mg, 0.5mg, 1.0 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1.0g, 2.5 g, 5 g, 7.5 g, 10 g, 25 g, 50 g, 75 g or 100 g per kilogram ofcomposition and 150 g, 100 g, 50 g, 40 g, 30 g, 20 g, 10 g, 7.5 g, 5 g,2.5 g, 1.0 g, 750 mg, 500 mg, 250 mg, 100 mg, 50 mg, 25 mg, 10 mg, 5 mg,2.5 mg or 1 mg per kilogram of composition, or any combination thereof.

In one embodiment to any part of the second aspect, the granulecomprises one or more formulating agents (such as those describedherein), preferably a formulating agent selected from the listconsisting of glycerol, ethylene glycol, 1, 2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate,sodium sulfate, potassium sulfate, magnesium sulfate, sodiumthiosulfate, calcium carbonate, sodium citrate, dextrin, glucose,sucrose, sorbitol, lactose, starch, kaolin, maltodextrin, cyclodextrin,wheat, PVA, acetate, phosphate and cellulose, preferably selected fromthe list consisting of 1, 2-propylene glycol, 1, 3-propylene glycol,sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin andcalcium carbonate.

In one embodiment to any part of the second aspect, the granulecomprises a core particle and one or more coatings. In a preferredembodiment, the coating comprises salt and/or wax and/or flour.Preferred formulations are disclosed in the formulation section below.

In one embodiment to any part of the second aspect, the granulecomprises one or more additional enzymes. The one or more additionalenzymes is preferably selected from the group consisting of acetyl xylanesterase, alpha-amylase, beta-amylase, arabinofuranosidase,cellobiohydrolases, cellulase, feruloyl esterase, galactanase,alpha-galactosidase, beta-galactosidase, beta-glucanase,beta-glucosidase, lipase, lysophospholipase, lysozyme, mannanase,alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1,phospholipase A2, phospholipase C, phospholipase D, protease,pullulanase, pectinase, pectin lyase, xylanase, beta-xylosidase or anycombination thereof.

In one embodiment to any part of the second aspect, the granulecomprises one or more probiotics. The one or more probiotics ispreferably selected from the group consisting of Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus,Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacteriumanimalis, Bifidobacterium sp., Carnobacterium sp., Clostridiumbutyricum, Clostridium sp., Enterococcus faecium, Enterococcus sp.,Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus farciminus,Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillussalivarius, Lactococcus lactis, Lactococcus sp., Leuconostoc sp.,Megasphaera elsdenii, Megasphaera sp., Pediococcus acidilactici,Pediococcus sp., Propionibacterium thoenii, Propionibacterium sp. andStreptococcus sp. or any combination thereof.

Liquid Formulations Comprising Polypeptides Having Lysozyme Activity

In a third aspect, the invention relates to liquid formulations, whereinthe liquid formulation comprises:

(a) 0.01% to 25% w/w of LYS polypeptide wherein:

-   -   (i) the LYS polypeptide has lysozyme activity;    -   (ii) the LYS polypeptide comprises one or more LAD catalytic        domains, wherein the LAD catalytic domain gives a domT score of        at least 170 when queried using a Profile Hidden Markov Model        prepared using SEQ ID NOs: 46 to 187 and hmmbuild software        program, and wherein the query is carried out using hmmscan        software program by the Method of Determining the LAD Catalytic        Domain by HMM;

(b) 20% to 80% w/w of polyol;

(c) 0.01% to 2.0% w/w preservative; and

(d) water.

In an embodiment, the domT score of the LAD catalytic domain is at least175, preferably at least 180, more preferably at least 185, even morepreferably at least 190, even more preferably at least 195, or mostpreferably at least 200. In an embodiment, the domT score of the LED isat least 103, preferably at least 106, more preferably at least 109,more preferably at least 112, more preferably at least 115, morepreferably at least 118, even more preferably at least 121, or mostpreferably at least 124. Preferred combinations of domT scores are asdisclosed in the first aspect of the invention.

In an embodiment, the polypeptide further comprises one or more lysozymeenhancing domains (LED). Thus, the invention further relates to a liquidformulation, wherein the liquid formulation comprises:

(a) 0.01% to 25% w/w of LYS polypeptide wherein:

-   -   (i) the LYS polypeptide has lysozyme activity;    -   (ii) the LYS polypeptide comprises one or more LAD catalytic        domains, wherein the LAD catalytic domain gives a domT score of        at least 170 when queried using a Profile Hidden Markov Model        prepared using SEQ ID NOs: 46 to 187 and hmmbuild software        program, and wherein the query is carried out using hmmscan        software program by the Method of Determining the LAD Catalytic        Domain by HMM;    -   (iii) the LYS polypeptide comprises one or more LED domains,        wherein the LED gives a domT score of at least 100 when queried        using a Profile Hidden Markov Model prepared using SEQ ID NOs:        188 to 316 and hmmbuild software program, and wherein the query        is carried out using the hmmscan software program by the Method        of Determining the Lysozyme Enhancing Domain by HMM;

(b) 20% to 80% w/w of polyol;

(c) 0.01% to 2.0% w/w preservative; and

(d) water.

In an embodiment, the domT score of the LAD catalytic domain is at least175, preferably at least 180, more preferably at least 185, even morepreferably at least 190, even more preferably at least 195, or mostpreferably at least 200. In an embodiment, the domT score of the LED isat least 103, preferably at least 106, more preferably at least 109,more preferably at least 112, more preferably at least 115, morepreferably at least 118, even more preferably at least 121, or mostpreferably at least 124. Preferred combinations of domT scores are asdisclosed in the first aspect of the invention.

In one embodiment of the third aspect, the invention relates to a liquidformulation comprising one or more LYS polypeptides having lysozymeactivity, wherein the liquid formulation comprises:

-   -   (A) 0.01% to 25% w/w of LYS polypeptide wherein the LYS        polypeptide is selected from the group consisting of:        -   (a) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 3;        -   (b) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 6;        -   (c) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 9;        -   (d) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 12;        -   (e) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 15;        -   (f) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 18;        -   (g) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 21;        -   (h) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 24;        -   (i) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 27;        -   (j) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 30;        -   (k) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 33;        -   (l) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 36;        -   (m) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 39;        -   (n) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 42;        -   (o) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to the            polypeptide of SEQ ID NO: 45;        -   (p) a variant of the polypeptide selected from the group            consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ            ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ            ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ            ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45,            wherein the variant has lysozyme activity and comprises one            or more amino acid substitutions, and/or one or more amino            acid deletions, and/or one or more amino acid insertions or            any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,            11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,            26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,            41, 42, 43, 44 or 45 positions;        -   (q) a polypeptide comprising the polypeptide of (a), (b),            (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m),            (n), (o) or (p) and a N-terminal and/or C-terminal His-tag            and/or HQ-tag;        -   (r) a polypeptide comprising the polypeptide of (a), (b),            (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m),            (n), (o) or (p) and a N-terminal and/or C-terminal extension            of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or            10 amino acids; and        -   (s) a fragment of the polypeptide of (a), (b), (c), (d),            (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p)            having lysozyme activity and having at least 90% of the            length of the mature polypeptide;    -   (B) 20% to 80% w/w of polyol;    -   (D) 0.01% to 2.0% w/w preservative; and    -   (D) water.

In one embodiment of the third aspect, the invention relates to a liquidformulation comprising one or more LYS polypeptides having lysozymeactivity, wherein the liquid formulation comprises:

-   -   (A) 0.01% to 25% w/w of LYS polypeptide wherein the LYS        polypeptide comprises a LAD catalytic domain that is selected        from the group consisting of:        -   (a) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 84 to 226 of SEQ ID NO: 3;        -   (b) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 84 to 226 of SEQ ID NO: 6;        -   (c) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 81 to 220 of SEQ ID NO: 9;        -   (d) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 161 to 304 of SEQ ID NO: 12;        -   (e) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 85 to 228 of SEQ ID NO: 15;        -   (f) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 88 to 230 of SEQ ID NO: 18;        -   (g) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 87 to 230 of SEQ ID NO: 21;        -   (h) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 90 to 232 of SEQ ID NO: 24;        -   (i) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 85 to 228 of SEQ ID NO: 27;        -   (j) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 85 to 228 of SEQ ID NO: 30;        -   (k) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 84 to 226 of SEQ ID NO: 33;        -   (l) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 83 to 222 of SEQ ID NO: 36;        -   (m) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 82 to 225 of SEQ ID NO: 39;        -   (n) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 161 to 303 of SEQ ID NO: 42; and        -   (o) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 85 to 227 of SEQ ID NO: 45;    -   (B) 20% to 80% w/w of polyol;    -   (C) 0.01% to 2.0% w/w preservative; and    -   (D) water.

In one embodiment of the third aspect, the invention relates to a liquidformulation comprising one or more LYS polypeptides having lysozymeactivity, wherein the liquid formulation comprises:

-   -   (A) 0.01% to 25% w/w of LYS polypeptide wherein the LYS        polypeptide comprises a LAD catalytic domain that is selected        from the group consisting of:        -   (a) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 84 to 226 of SEQ ID NO: 3;        -   (b) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 84 to 226 of SEQ ID NO: 6;        -   (c) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 81 to 220 of SEQ ID NO: 9;        -   (d) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 161 to 304 of SEQ ID NO: 12;        -   (e) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 85 to 228 of SEQ ID NO: 15;        -   (f) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 88 to 230 of SEQ ID NO: 18;        -   (g) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 87 to 230 of SEQ ID NO: 21;        -   (h) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 90 to 232 of SEQ ID NO: 24;        -   (i) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 85 to 228 of SEQ ID NO: 27;        -   (j) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 85 to 228 of SEQ ID NO: 30;        -   (k) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 84 to 226 of SEQ ID NO: 33;        -   (l) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 83 to 222 of SEQ ID NO: 36;        -   (m) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 82 to 225 of SEQ ID NO: 39;        -   (n) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 161 to 303 of SEQ ID NO: 42; and        -   (o) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 85 to 227 of SEQ ID NO: 45;    -   (B) the LYS polypeptide comprises a LED domain selected from the        group consisting of:        -   (a) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 3;        -   (b) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 6;        -   (c) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 9;        -   (d) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 72 of SEQ ID NO: 12;        -   (e) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 15;        -   (f) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 18;        -   (g) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 21;        -   (h) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 24;        -   (i) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 27;        -   (j) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 30;        -   (k) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 33;        -   (l) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 36;        -   (m) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 72 of SEQ ID NO: 39;        -   (n) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 42;        -   (o) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 1 to 73 of SEQ ID NO: 45;        -   (d) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 96 to 167 of SEQ ID NO: 12; and        -   (n) a polypeptide having at least 80%, e.g., at least 85%,            at least 90% or at least 95% sequence identity to amino            acids 96 to 168 of SEQ ID NO: 42;    -   (C) 20% to 80% w/w of polyol;    -   (D) 0.01% to 2.0% w/w preservative; and    -   (E) water.

In one embodiment to any part of the third aspect, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317). In an embodiment, the LAD catalytic domain comprises one ormore motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, theLED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif I:AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317) and the LED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or moremotif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:319). In an embodiment, the LAD catalytic domain comprises one or moremotif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or moremotif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises oneor more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ IDNO: 319).

In one embodiment to any part of the third aspect, the polypeptide is offungal origin. In an embodiment, the polypeptide is obtained orobtainable from the taxonomic phylum Ascomycota, preferably thetaxonomic subphylum Pezizomycotina.

In one embodiment to any part of the third aspect, the liquidformulation comprises one or more formulating agents (such as thosedescribed herein), preferably a formulating agent selected from the listconsisting of glycerol, ethylene glycol, 1, 2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate,sodium sulfate, potassium sulfate, magnesium sulfate, sodiumthiosulfate, calcium carbonate, sodium citrate, dextrin, glucose,sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate,preferably selected from the list consisting of 1, 2-propylene glycol,1, 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodiumthiosulfate, kaolin and calcium carbonate.

In one embodiment to any part of the third aspect, the liquidformulation comprises one or more polyols, preferably a polyol selectedfrom the group consisting of glycerol, sorbitol, propylene glycol (MPG),ethylene glycol, diethylene glycol, triethylene glycol, 1, 2-propyleneglycol or 1, 3-propylene glycol, dipropylene glycol, polyethylene glycol(PEG) having an average molecular weight below about 600 andpolypropylene glycol (PPG) having an average molecular weight belowabout 600, more preferably selected from the group consisting ofglycerol, sorbitol and propylene glycol (MPG) or any combinationthereof.

In one embodiment to any part of the third aspect, the liquidformulation comprises 20%-80% polyol (i.e. total amount of polyol),preferably 25%-75% polyol, more preferably 30%-70% polyol, morepreferably 35%-65% polyol or most preferably 40%-60% polyol. In oneembodiment to any part of the third aspect, the liquid formulationcomprises 20%-80% polyol, preferably 25%-75% polyol, more preferably30%-70% polyol, more preferably 35%-65% polyol or most preferably40%-60% polyol wherein the polyol is selected from the group consistingof glycerol, sorbitol, propylene glycol (MPG), ethylene glycol,diethylene glycol, triethylene glycol, 1, 2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) havingan average molecular weight below about 600 and polypropylene glycol(PPG) having an average molecular weight below about 600. In oneembodiment to any part of the third aspect, the liquid formulationcomprises 20%-80% polyol (i.e. total amount of polyol), preferably25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65%polyol or most preferably 40%-60% polyol wherein the polyol is selectedfrom the group consisting of glycerol, sorbitol and propylene glycol(MPG).

In one embodiment to any part of the third aspect, the preservative isselected from the group consisting of sodium sorbate, potassium sorbate,sodium benzoate and potassion benzoate or any combination thereof. Inone embodiment, the liquid formulation comprises 0.02% to 1.5% w/wpreservative, more preferably 0.05% to 1.0% w/w preservative or mostpreferably 0.1% to 0.5% w/w preservative. In one embodiment, the liquidformulation comprises 0.01% to 2.0% w/w preservative (i.e. total amountof preservative), preferably 0.02% to 1.5% w/w preservative, morepreferably 0.05% to 1.0% w/w preservative or most preferably 0.1% to0.5% w/w preservative wherein the preservative is selected from thegroup consisting of sodium sorbate, potassium sorbate, sodium benzoateand potassion benzoate or any combination thereof.

In one embodiment to any part of the third aspect, the liquidformulation comprises 0.05% to 20% w/w LYS polypeptide, more preferably0.2% to 15% w/w LYS polypeptide, more preferably 0.5% to 15% w/w LYSpolypeptide or most preferably 1.0% to 10% w/w LYS polypeptide.

In one embodiment to any part of the third aspect, the liquidformulation comprises one or more additional enzymes. The one or moreadditional enzymes is preferably selected from the group consisting ofacetyl xylan esterase, alpha-amylase, beta-amylase, arabinofuranosidase,cellobiohydrolases, cellulase, feruloyl esterase, galactanase,alpha-galactosidase, beta-galactosidase, beta-glucanase,beta-glucosidase, lipase, lysophospholipase, lysozyme, mannanase,alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1,phospholipase A2, phospholipase C, phospholipase D, protease,pullulanase, pectinase, pectin lyase, xylanase, beta-xylosidase or anycombination thereof.

In one embodiment to any part of the third aspect, the liquidformulation comprises one or more probiotics. The one or more probioticsis preferably selected from the group consisting of Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus,Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacteriumanimalis, Bifidobacterium sp., Carnobacterium sp., Clostridiumbutyricum, Clostridium sp., Enterococcus faecium, Enterococcus sp.,Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus farciminus,Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillussalivarius, Lactococcus lactis, Lactococcus sp., Leuconostoc sp.,Megasphaera elsdenii, Megasphaera sp., Pediococcus acidilactici,Pediococcus sp., Propionibacterium thoenii, Propionibacterium sp. andStreptococcus sp. or any combination thereof.

Polypeptides Having Lysozyme Activity

In a fourth aspect, the invention relates to polypeptides havinglysozyme activity having at least 95%, e.g., at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 2. In one embodiment, the polypeptides differby up to 11 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11amino acids from the mature polypeptide of SEQ ID NO: 2.

In a continuation of the fourth aspect, the invention relates topolypeptides having lysozyme activity having at least 95%, e.g., atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 3. In one embodiment, the polypeptides differ byup to 11 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 aminoacids from SEQ ID NO: 3.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 3 of at least 95%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 3. In one embodiment, lysozyme activity is determined asdescribed in example 1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 3; comprises the amino acid sequence of SEQ ID NO: 3 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 3 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 3. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 226 of SEQ ID NO:3. In one embodiment, the polypeptide has been isolated.

In a continuation of the fourth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 95%, e.g., at least 96%, at least 97%, at least 98%,at least 99%, or 100%. In a further embodiment, the polypeptide has beenisolated.

In a continuation of the fourth aspect, the invention relates tovariants of SEQ ID NO: 3 having lysozyme activity comprising one or moreamino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 3 is not more than 11, e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or 11. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 3 is not more than 10, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number ofsubstitutions and/or deletions and/or insertions in SEQ ID NO: 3 is notmore than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a furtherembodiment, the number of substitutions, preferably conservativesubstitutions, in SEQ ID NO: 3 is not more than 10, e.g. 1, 2, 3, 4, 5,6, 7, 8, 9, or 10.

In an embodiment of the fourth aspect, the variant has at least 50%,such as at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 3. In one embodiment, lysozyme activityis determined as described in example 1.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly. Other examples of conservativesubstitutions are G to A; A to G, S; V to I, L, A, T, S; I to V, L, M; Lto I, M, V; M to L, I, V; P to A, S, N; F to Y, W, H; Y to F, W, H; W toY, F, H; R to K, E, D; K to R, E, D; H to Q, N, S; D to N, E, K, R, Q; Eto Q, D, K, R, N; S to T, A; T to S, V, A; C to S, T, A; N to D, Q, H,S; Q to E, N, H, K, R.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for lysozyme activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labelling, in conjunction with mutation of putativecontact site amino acids. See, for example, de Vos et al., 1992, Science255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaveret al., 1992, FEBS Lett. 309: 59-64. The identity of essential aminoacids can also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide or a fusion polypeptide.

In a fifth aspect, the invention relates to polypeptides having lysozymeactivity having at least 94%, e.g., at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 5. In one embodiment, the polypeptides differby up to 13 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or13 amino acids from the mature polypeptide of SEQ ID NO: 5.

In a continuation of the fifth aspect, the invention relates topolypeptides having lysozyme activity having at least 94%, e.g., atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 6. In one embodiment, thepolypeptides differ by up to 13 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, or 13 amino acids from SEQ ID NO: 6.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 6 of at least 94%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 6. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 6of at least 95% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 6. In one embodiment, lysozyme activity isdetermined as described in example 1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 5. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 6; comprises the amino acid sequence of SEQ ID NO: 6 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 6 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 6. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 226 of SEQ ID NO:6. In one embodiment, the polypeptide has been isolated.

In a continuation of the fifth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 4 of at least 94%, e.g., at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100%. In a further embodiment, thepolypeptide has been isolated.

In a continuation of the fifth aspect, the invention relates to variantsof SEQ ID NO: 6 having lysozyme activity comprising one or more aminoacid substitutions, and/or one or more amino acid deletions, and/or oneor more amino acid insertions or any combination thereof at one or more(e.g., several) positions. In an embodiment, the number of positionscomprising one or more amino acid substitutions, and/or one or moreamino acid deletions, and/or one or more amino acid insertions or anycombination thereof in SEQ ID NO: 6 is not more than 13, e.g. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 6 is not more than 10, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number ofsubstitutions and/or deletions and/or insertions in SEQ ID NO: 6 is notmore than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a furtherembodiment, the number of substitutions, preferably conservativesubstitutions, in SEQ ID NO: 6 is not more than 10, e.g. 1, 2, 3, 4, 5,6, 7, 8, 9, or 10. Examples of amino acid changes and conservativesubstitutions are described in the fourth aspect of the invention.

In an embodiment of the fifth aspect, the variant has at least 50%, suchas at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 6. In one embodiment, lysozyme activityis determined as described in example 1.

In a sixth aspect, the invention relates to polypeptides having lysozymeactivity having at least 80%, e.g., at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to the mature polypeptideof SEQ ID NO: 8. In one embodiment, the polypeptides differ by up to 44amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids from themature polypeptide of SEQ ID NO: 8.

In a continuation of the sixth aspect, the invention relates topolypeptides having lysozyme activity having at least 80%, e.g., atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 9. In one embodiment, the polypeptides differ byup to 44 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids fromSEQ ID NO: 9.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 9 of at least 80%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 9. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 9of at least 85% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 9. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 9 of at least 90% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 9. In one embodiment, theinvention relates to polypeptides having lysozyme activity and having asequence identity to SEQ ID NO: 9 of at least 95% and wherein thepolypeptide has at least 50%, such as at least 75%, at least 90%, atleast 95% or at least 100% of the lysozyme activity of SEQ ID NO: 9. Inone embodiment, lysozyme activity is determined as described in example1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 8. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 9; comprises the amino acid sequence of SEQ ID NO: 9 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 9 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 9. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 223 of SEQ ID NO:9. In one embodiment, the polypeptide has been isolated.

In a continuation of the sixth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 7 of at least 80%, e.g., at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In a further embodiment, the polypeptide has beenisolated.

In a continuation of the sixth aspect, the invention relates to variantsof SEQ ID NO: 9 having lysozyme activity comprising one or more aminoacid substitutions, and/or one or more amino acid deletions, and/or oneor more amino acid insertions or any combination thereof at one or more(e.g., several) positions. In an embodiment, the number of positionscomprising one or more amino acid substitutions, and/or one or moreamino acid deletions, and/or one or more amino acid insertions or anycombination thereof in SEQ ID NO: 9 is not more than 44, e.g. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, or 44. In an embodiment, the number of positions comprisingone or more amino acid substitutions, and/or one or more amino aciddeletions, and/or one or more amino acid insertions or any combinationthereof in SEQ ID NO: 9 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9 or 10. In one embodiment, the number of substitutions and/ordeletions and/or insertions in SEQ ID NO: 9 is not more than 10, e.g. 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number ofsubstitutions, preferably conservative substitutions, in SEQ ID NO: 9 isnot more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples ofamino acid changes and conservative substitutions are described in thefourth aspect of the invention.

In an embodiment of the sixth aspect, the variant has at least 50%, suchas at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 9. In one embodiment, lysozyme activityis determined as described in example 1.

In a seventh aspect, the invention relates to polypeptides havinglysozyme activity having at least 80%, e.g., at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 11. In one embodiment, the polypeptides differby up to 50 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 amino acids from the mature polypeptide of SEQ ID NO: 11.

In a continuation of the seventh aspect, the invention relates topolypeptides having lysozyme activity having at least 80%, e.g., atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 12. In one embodiment, the polypeptides differ byup to 50 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 amino acids from SEQ ID NO: 12.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 12 of at least 80%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 12. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 12of at least 85% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 12. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 12 of at least 90% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 12. In one embodiment, theinvention relates to polypeptides having lysozyme activity and having asequence identity to SEQ ID NO: 12 of at least 95% and wherein thepolypeptide has at least 50%, such as at least 75%, at least 90%, atleast 95% or at least 100% of the lysozyme activity of SEQ ID NO: 12. Inone embodiment, lysozyme activity is determined as described in example1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 11. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 12; comprises the amino acid sequence of SEQ ID NO: 12 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 12 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 12. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 304 of SEQ ID NO:12. In one embodiment, the polypeptide has been isolated.

In a continuation of the seventh aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 10 of at least 80%, e.g., at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In a further embodiment, the polypeptide has beenisolated.

In a continuation of the seventh aspect, the invention relates tovariants of SEQ ID NO: 12 having lysozyme activity comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 12 is not more than 50, e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In an embodiment, the numberof positions comprising one or more amino acid substitutions, and/or oneor more amino acid deletions, and/or one or more amino acid insertionsor any combination thereof in SEQ ID NO: 12 is not more than 10, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number ofsubstitutions and/or deletions and/or insertions in SEQ ID NO: 12 is notmore than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a furtherembodiment, the number of substitutions, preferably conservativesubstitutions, in SEQ ID NO: 12 is not more than 10, e.g. 1, 2, 3, 4, 5,6, 7, 8, 9, or 10. Examples of amino acid changes and conservativesubstitutions are described in the fourth aspect of the invention.

In an embodiment of the seventh aspect, the variant has at least 50%,such as at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 12. In one embodiment, lysozyme activityis determined as described in example 1.

In a eighth aspect, the invention relates to polypeptides havinglysozyme activity having at least 87%, e.g., at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to the mature polypeptide of SEQ ID NO: 14. Inone embodiment, the polypeptides differ by up to 29 amino acids, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids from the maturepolypeptide of SEQ ID NO: 14.

In a continuation of the eighth aspect, the invention relates topolypeptides having lysozyme activity having at least 87%, e.g., atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15. Inone embodiment, the polypeptides differ by up to 29 amino acids, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids from SEQ ID NO: 15.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 15 of at least 87%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 15. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 15of at least 90% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 15. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 15 of at least 95% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 15. In one embodiment, lysozymeactivity is determined as described in example 1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 14. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 15; comprises the amino acid sequence of SEQ ID NO: 15 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 15 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 15. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 228 of SEQ ID NO:15. In one embodiment, the polypeptide has been isolated.

In a continuation of the eighth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 13 of at least 87%, e.g., at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%. In afurther embodiment, the polypeptide has been isolated.

In a continuation of the eighth aspect, the invention relates tovariants of SEQ ID NO: 15 having lysozyme activity comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 15 is not more than 29, e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, or 29. In an embodiment, the number of positionscomprising one or more amino acid substitutions, and/or one or moreamino acid deletions, and/or one or more amino acid insertions or anycombination thereof in SEQ ID NO: 15 is not more than 10, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutionsand/or deletions and/or insertions in SEQ ID NO: 15 is not more than 10,e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, thenumber of substitutions, preferably conservative substitutions, in SEQID NO: 15 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of amino acid changes and conservative substitutions aredescribed in the fourth aspect of the invention.

In an embodiment of the eighth aspect, the variant has at least 50%,such as at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 15. In one embodiment, lysozyme activityis determined as described in example 1.

In a ninth aspect, the invention relates to polypeptides having lysozymeactivity having at least 81%, e.g., at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:17. In one embodiment, the polypeptides differ by up to 43 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, or 43 amino acids from the mature polypeptide of SEQID NO: 17.

In a continuation of the ninth aspect, the invention relates topolypeptides having lysozyme activity having at least 81%, e.g., atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 18. In one embodiment, the polypeptides differ by up to 43 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, or 43 amino acids from SEQ ID NO: 18.

In a continuation of the ninth aspect, the invention relates topolypeptides having lysozyme activity having at least 81%, e.g., atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 18. In one embodiment, the polypeptides differ by up to 28 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 amino acids from SEQ ID NO:239.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 18 of at least 81%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 18. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 18of at least 85% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 18. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 18 of at least 90% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 18. In one embodiment, theinvention relates to polypeptides having lysozyme activity and having asequence identity to SEQ ID NO: 18 of at least 95% and wherein thepolypeptide has at least 50%, such as at least 75%, at least 90%, atleast 95% or at least 100% of the lysozyme activity of SEQ ID NO: 18. Inone embodiment, lysozyme activity is determined as described in example1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 17. In one embodiment, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 239. Inone embodiment, the polypeptide preferably comprises or consists of theamino acid sequence of SEQ ID NO: 18; comprises the amino acid sequenceof SEQ ID NO: 18 and a N-terminal and/or C-terminal His-tag and/orHQ-tag; comprises the amino acid sequence of SEQ ID NO: 18 and aN-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or is a fragment thereofhaving lysozyme activity and having at least 90% such as at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% of the length of SEQ ID NO: 18.In one embodiment, the polypeptide comprises or consists of amino acids1 to 230 of SEQ ID NO: 18. In one embodiment, the polypeptide comprisesor consists of amino acids 1 to 146 of SEQ ID NO: 18. In one embodiment,the polypeptide has been isolated.

In a continuation of the ninth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 16 of at least 81%, e.g., at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. In a further embodiment, the polypeptide has been isolated.

In a continuation of the ninth aspect, the invention relates to variantsof SEQ ID NO: 18 having lysozyme activity comprising one or more aminoacid substitutions, and/or one or more amino acid deletions, and/or oneor more amino acid insertions or any combination thereof at one or more(e.g., several) positions. In an embodiment, the number of positionscomprising one or more amino acid substitutions, and/or one or moreamino acid deletions, and/or one or more amino acid insertions or anycombination thereof in SEQ ID NO: 18 is not more than 43, e.g. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, or 43. In an embodiment, the number of positions comprising oneor more amino acid substitutions, and/or one or more amino aciddeletions, and/or one or more amino acid insertions or any combinationthereof in SEQ ID NO: 18 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9 or 10. In one embodiment, the number of substitutions and/ordeletions and/or insertions in SEQ ID NO: 18 is not more than 10, e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number ofsubstitutions, preferably conservative substitutions, in SEQ ID NO: 18is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples ofamino acid changes and conservative substitutions are described in thefourth aspect of the invention.

In an embodiment of the ninth aspect, the variant has at least 50%, suchas at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 18. In one embodiment, lysozyme activityis determined as described in example 1.

In a tenth aspect, the invention relates to polypeptides having lysozymeactivity having at least 80%, e.g., at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to the mature polypeptideof SEQ ID NO: 20. In one embodiment, the polypeptides differ by up to 45amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids from themature polypeptide of SEQ ID NO: 20.

In a continuation of the tenth aspect, the invention relates topolypeptides having lysozyme activity having at least 80%, e.g., atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 21. In one embodiment, the polypeptides differ byup to 45 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acidsfrom SEQ ID NO: 21.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 21 of at least 80%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 21. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 21of at least 85% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 21. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 21 of at least 90% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 21. In one embodiment, theinvention relates to polypeptides having lysozyme activity and having asequence identity to SEQ ID NO: 21 of at least 95% and wherein thepolypeptide has at least 50%, such as at least 75%, at least 90%, atleast 95% or at least 100% of the lysozyme activity of SEQ ID NO: 21. Inone embodiment, lysozyme activity is determined as described in example1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 20. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 21; comprises the amino acid sequence of SEQ ID NO: 21 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 21 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 21. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 230 of SEQ ID NO:21. In one embodiment, the polypeptide has been isolated.

In a continuation of the tenth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 19 of at least 80%, e.g., at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In a further embodiment, the polypeptide has beenisolated.

In a continuation of the tenth aspect, the invention relates to variantsof SEQ ID NO: 21 having lysozyme activity comprising one or more aminoacid substitutions, and/or one or more amino acid deletions, and/or oneor more amino acid insertions or any combination thereof at one or more(e.g., several) positions. In an embodiment, the number of positionscomprising one or more amino acid substitutions, and/or one or moreamino acid deletions, and/or one or more amino acid insertions or anycombination thereof in SEQ ID NO: 21 is not more than 45, e.g. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, or 45. In an embodiment, the number of positionscomprising one or more amino acid substitutions, and/or one or moreamino acid deletions, and/or one or more amino acid insertions or anycombination thereof in SEQ ID NO: 21 is not more than 10, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutionsand/or deletions and/or insertions in SEQ ID NO: 21 is not more than 10,e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, thenumber of substitutions, preferably conservative substitutions, in SEQID NO: 21 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of amino acid changes and conservative substitutions aredescribed in the fourth aspect of the invention.

In an embodiment of the tenth aspect, the variant has at least 50%, suchas at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 21. In one embodiment, lysozyme activityis determined as described in example 1.

In a eleventh aspect, the invention relates to polypeptides havinglysozyme activity having at least 80%, e.g., at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 23. In one embodiment, the polypeptides differby up to 46 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 aminoacids from the mature polypeptide of SEQ ID NO: 23.

In a continuation of the eleventh aspect, the invention relates topolypeptides having lysozyme activity having at least 80%, e.g., atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 24. In one embodiment, the polypeptides differ byup to 46 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 aminoacids from SEQ ID NO: 24.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 24 of at least 80%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 24. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 24of at least 85% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 24. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 24 of at least 90% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 24. In one embodiment, theinvention relates to polypeptides having lysozyme activity and having asequence identity to SEQ ID NO: 24 of at least 95% and wherein thepolypeptide has at least 50%, such as at least 75%, at least 90%, atleast 95% or at least 100% of the lysozyme activity of SEQ ID NO: 24. Inone embodiment, lysozyme activity is determined as described in example1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 23. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 24; comprises the amino acid sequence of SEQ ID NO: 24 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 24 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 24. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 232 of SEQ ID NO:24. In one embodiment, the polypeptide has been isolated.

In a continuation of the eleventh aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 22 of at least 80%, e.g., at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In a further embodiment, the polypeptide has beenisolated.

In a continuation of the eleventh aspect, the invention relates tovariants of SEQ ID NO: 24 having lysozyme activity comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 24 is not more than 46, e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, or 46. In an embodiment, the number of positionscomprising one or more amino acid substitutions, and/or one or moreamino acid deletions, and/or one or more amino acid insertions or anycombination thereof in SEQ ID NO: 24 is not more than 10, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutionsand/or deletions and/or insertions in SEQ ID NO: 24 is not more than 10,e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, thenumber of substitutions, preferably conservative substitutions, in SEQID NO: 24 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of amino acid changes and conservative substitutions aredescribed in the fourth aspect of the invention.

In an embodiment of the eleventh aspect, the variant has at least 50%,such as at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 24. In one embodiment, lysozyme activityis determined as described in example 1.

In a twelfth aspect, the invention relates to polypeptides havinglysozyme activity having at least 87%, e.g., at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to the mature polypeptide of SEQ ID NO: 26. Inone embodiment, the polypeptides differ by up to 29 amino acids, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids from the maturepolypeptide of SEQ ID NO: 26.

In a continuation of the twelfth aspect, the invention relates topolypeptides having lysozyme activity having at least 87%, e.g., atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27. Inone embodiment, the polypeptides differ by up to 29 amino acids, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids from SEQ ID NO: 27.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 27 of at least 87%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 27. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 27of at least 90% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 27. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 27 of at least 95% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 27. In one embodiment, lysozymeactivity is determined as described in example 1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 26. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 27; comprises the amino acid sequence of SEQ ID NO: 27 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 27 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 27. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 228 of SEQ ID NO:27. In one embodiment, the polypeptide has been isolated.

In a continuation of the twelfth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 25 of at least 87%, e.g., at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%. In afurther embodiment, the polypeptide has been isolated.

In a continuation of the twelfth aspect, the invention relates tovariants of SEQ ID NO: 27 having lysozyme activity comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 27 is not more than 29, e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, or 29. In an embodiment, the number of positionscomprising one or more amino acid substitutions, and/or one or moreamino acid deletions, and/or one or more amino acid insertions or anycombination thereof in SEQ ID NO: 27 is not more than 10, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutionsand/or deletions and/or insertions in SEQ ID NO: 27 is not more than 10,e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, thenumber of substitutions, preferably conservative substitutions, in SEQID NO: 27 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of amino acid changes and conservative substitutions aredescribed in the fourth aspect of the invention.

In an embodiment of the twelfth aspect, the variant has at least 50%,such as at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 27. In one embodiment, lysozyme activityis determined as described in example 1.

In a thirteenth aspect, the invention relates to polypeptides havinglysozyme activity having at least 96%, e.g., at least 97%, at least 98%,at least 99%, or 100% sequence identity to the mature polypeptide of SEQID NO: 29. In one embodiment, the polypeptides differ by up to 8 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from the maturepolypeptide of SEQ ID NO: 29.

In a continuation of the thirteenth aspect, the invention relates topolypeptides having lysozyme activity having at least 96%, e.g., atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 30. In one embodiment, the polypeptides differ by up to 8 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from SEQ ID NO: 30.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 30 of at least 96%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 30. In one embodiment, lysozyme activity is determined asdescribed in example 1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 29. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 30; comprises the amino acid sequence of SEQ ID NO: 30 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 30 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 30. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 228 of SEQ ID NO:30. In one embodiment, the polypeptide has been isolated.

In a continuation of the thirteenth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 28 of at least 96%, e.g., at least 97%, at least 98%, at least 99%,or 100%. In a further embodiment, the polypeptide has been isolated.

In a continuation of the thirteenth aspect, the invention relates tovariants of SEQ ID NO: 30 having lysozyme activity comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 30 is not more than 8, e.g. 1, 2,3, 4, 5, 6, 7, or 8. In one embodiment, the number of substitutionsand/or deletions and/or insertions in SEQ ID NO: 30 is not more than 8,e.g. 1, 2, 3, 4, 5, 6, 7, or 8. In a further embodiment, the number ofsubstitutions, preferably conservative substitutions, in SEQ ID NO: 30is not more than 8, e.g. 1, 2, 3, 4, 5, 6, 7, or 8. Examples of aminoacid changes and conservative substitutions are described in the fourthaspect of the invention.

In an embodiment of the thirteenth aspect, the variant has at least 50%,such as at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 30. In one embodiment, lysozyme activityis determined as described in example 1.

In a fourteenth aspect, the invention relates to polypeptides havinglysozyme activity having at least 80%, e.g., at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 32. In one embodiment, the polypeptides differby up to 45 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 aminoacids from the mature polypeptide of SEQ ID NO: 32.

In a continuation of the fourteenth aspect, the invention relates topolypeptides having lysozyme activity having at least 80%, e.g., atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 33. In one embodiment, the polypeptides differ byup to 45 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acidsfrom SEQ ID NO: 33.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 33 of at least 80%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 33. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 33of at least 85% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 33. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 33 of at least 90% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 33. In one embodiment, theinvention relates to polypeptides having lysozyme activity and having asequence identity to SEQ ID NO: 33 of at least 95% and wherein thepolypeptide has at least 50%, such as at least 75%, at least 90%, atleast 95% or at least 100% of the lysozyme activity of SEQ ID NO: 33. Inone embodiment, lysozyme activity is determined as described in example1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 32. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 33; comprises the amino acid sequence of SEQ ID NO: 33 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 33 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 33. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 226 of SEQ ID NO:33. In one embodiment, the polypeptide has been isolated.

In a continuation of the fourteenth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 31 of at least 80%, e.g., at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In a further embodiment, the polypeptide has beenisolated.

In a continuation of the fourteenth aspect, the invention relates tovariants of SEQ ID NO: 33 having lysozyme activity comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 33 is not more than 45, e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, or 45. In an embodiment, the number of positionscomprising one or more amino acid substitutions, and/or one or moreamino acid deletions, and/or one or more amino acid insertions or anycombination thereof in SEQ ID NO: 33 is not more than 10, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number of substitutionsand/or deletions and/or insertions in SEQ ID NO: 33 is not more than 10,e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, thenumber of substitutions, preferably conservative substitutions, in SEQID NO: 33 is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of amino acid changes and conservative substitutions aredescribed in the fourth aspect of the invention.

In an embodiment of the fourteenth aspect, the variant has at least 50%,such as at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 33. In one embodiment, lysozyme activityis determined as described in example 1.

In a fifteenth aspect, the invention relates to polypeptides havinglysozyme activity having at least 80%, e.g., at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 35. In one embodiment, the polypeptides differby up to 44 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acidsfrom the mature polypeptide of SEQ ID NO: 35.

In a continuation of the fifteenth aspect, the invention relates topolypeptides having lysozyme activity having at least 80%, e.g., atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 36. In one embodiment, the polypeptides differ byup to 44 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids fromSEQ ID NO: 36.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 36 of at least 80%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 36. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 36of at least 85% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 36. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 36 of at least 90% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 36. In one embodiment, theinvention relates to polypeptides having lysozyme activity and having asequence identity to SEQ ID NO: 36 of at least 95% and wherein thepolypeptide has at least 50%, such as at least 75%, at least 90%, atleast 95% or at least 100% of the lysozyme activity of SEQ ID NO: 36. Inone embodiment, lysozyme activity is determined as described in example1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 35. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 36; comprises the amino acid sequence of SEQ ID NO: 36 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 36 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 36. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 225 of SEQ ID NO:36. In one embodiment, the polypeptide has been isolated.

In a continuation of the fifteenth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 34 of at least 80%, e.g., at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In a further embodiment, the polypeptide has beenisolated.

In a continuation of the fifteenth aspect, the invention relates tovariants of SEQ ID NO: 36 having lysozyme activity comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 36 is not more than 44, e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, or 44. In an embodiment, the number of positions comprisingone or more amino acid substitutions, and/or one or more amino aciddeletions, and/or one or more amino acid insertions or any combinationthereof in SEQ ID NO: 36 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9 or 10. In one embodiment, the number of substitutions and/ordeletions and/or insertions in SEQ ID NO: 36 is not more than 10, e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, the number ofsubstitutions, preferably conservative substitutions, in SEQ ID NO: 36is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples ofamino acid changes and conservative substitutions are described in thefourth aspect of the invention.

In an embodiment of the fifteenth aspect, the variant has at least 50%,such as at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 36. In one embodiment, lysozyme activityis determined as described in example 1.

In a sixteenth aspect, the invention relates to polypeptides havinglysozyme activity having at least 81%, e.g., at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide of SEQ IDNO: 38. In one embodiment, the polypeptides differ by up to 42 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, or 42 amino acids from the mature polypeptide ofSEQ ID NO: 38.

In a continuation of the sixteenth aspect, the invention relates topolypeptides having lysozyme activity having at least 81%, e.g., atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 39. In one embodiment, the polypeptides differ by up to 42 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, or 42 amino acids from SEQ ID NO: 39.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 39 of at least 81%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 39. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 39of at least 85% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 39. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 39 of at least 90% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 39. In one embodiment, theinvention relates to polypeptides having lysozyme activity and having asequence identity to SEQ ID NO: 39 of at least 95% and wherein thepolypeptide has at least 50%, such as at least 75%, at least 90%, atleast 95% or at least 100% of the lysozyme activity of SEQ ID NO: 39. Inone embodiment, lysozyme activity is determined as described in example1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 38. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 39; comprises the amino acid sequence of SEQ ID NO: 39 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 39 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 39. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 225 of SEQ ID NO:39. In one embodiment, the polypeptide has been isolated.

In a continuation of the sixteenth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 37 of at least 81%, e.g., at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. In a further embodiment, the polypeptide has been isolated.

In a continuation of the sixteenth aspect, the invention relates tovariants of SEQ ID NO: 39 having lysozyme activity comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 39 is not more than 42, e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, or 42. In an embodiment, the number of positions comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof inSEQ ID NO: 39 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or10. In one embodiment, the number of substitutions and/or deletionsand/or insertions in SEQ ID NO: 39 is not more than 10, e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, or 10. In a further embodiment, the number ofsubstitutions, preferably conservative substitutions, in SEQ ID NO: 39is not more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples ofamino acid changes and conservative substitutions are described in thefourth aspect of the invention.

In an embodiment of the sixteenth aspect, the variant has at least 50%,such as at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 39. In one embodiment, lysozyme activityis determined as described in example 1.

In a seventeenth aspect, the invention relates to polypeptides havinglysozyme activity having at least 80%, e.g., at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 41. In one embodiment, the polypeptides differby up to 50 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 amino acids from the mature polypeptide of SEQ ID NO: 41.

In a continuation of the seventeenth aspect, the invention relates topolypeptides having lysozyme activity having at least 80%, e.g., atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 42. In one embodiment, the polypeptides differ byup to 50 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 amino acids from SEQ ID NO: 42.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 42 of at least 80%and wherein the polypeptide has at least 50%, such as at least 75%, atleast 90%, at least 95% or at least 100% of the lysozyme activity of SEQID NO: 42. In one embodiment, the invention relates to polypeptideshaving lysozyme activity and having a sequence identity to SEQ ID NO: 42of at least 85% and wherein the polypeptide has at least 50%, such as atleast 75%, at least 90%, at least 95% or at least 100% of the lysozymeactivity of SEQ ID NO: 42. In one embodiment, the invention relates topolypeptides having lysozyme activity and having a sequence identity toSEQ ID NO: 42 of at least 90% and wherein the polypeptide has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 42. In one embodiment, theinvention relates to polypeptides having lysozyme activity and having asequence identity to SEQ ID NO: 42 of at least 95% and wherein thepolypeptide has at least 50%, such as at least 75%, at least 90%, atleast 95% or at least 100% of the lysozyme activity of SEQ ID NO: 42. Inone embodiment, lysozyme activity is determined as described in example1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 41. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 42; comprises the amino acid sequence of SEQ ID NO: 42 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 42 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 42. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 304 of SEQ ID NO:42. In one embodiment, the polypeptide has been isolated.

In a continuation of the seventeenth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 40 of at least 80%, e.g., at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In a further embodiment, the polypeptide has beenisolated.

In a continuation of the seventeenth aspect, the invention relates tovariants of SEQ ID NO: 42 having lysozyme activity comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 42 is not more than 50, e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In an embodiment, the numberof positions comprising one or more amino acid substitutions, and/or oneor more amino acid deletions, and/or one or more amino acid insertionsor any combination thereof in SEQ ID NO: 42 is not more than 10, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the number ofsubstitutions and/or deletions and/or insertions in SEQ ID NO: 42 is notmore than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a furtherembodiment, the number of substitutions, preferably conservativesubstitutions, in SEQ ID NO: 42 is not more than 10, e.g. 1, 2, 3, 4, 5,6, 7, 8, 9, or 10. Examples of amino acid changes and conservativesubstitutions are described in the fourth aspect of the invention.

In an embodiment of the seventeenth aspect, the variant has at least50%, such as at least 75%, at least 90%, at least 95% or at least 100%of the lysozyme activity of SEQ ID NO: 42. In one embodiment, lysozymeactivity is determined as described in example 1.

In a eighteenth aspect, the invention relates to polypeptides havinglysozyme activity having at least 100%, e.g., or 100% sequence identityto the mature polypeptide of SEQ ID NO: 44. In one embodiment, thepolypeptides differ by up to 0 amino acids, e.g., or 1 amino acids fromthe mature polypeptide of SEQ ID NO: 44.

In a continuation of the eighteenth aspect, the invention relates topolypeptides having lysozyme activity having at least 100%, e.g., or100% sequence identity to SEQ ID NO: 45. In one embodiment, thepolypeptides differ by up to 0 amino acids, e.g., or 1 amino acids fromSEQ ID NO: 45.

In one embodiment, the invention relates to polypeptides having lysozymeactivity and having a sequence identity to SEQ ID NO: 45 of at least100% and wherein the polypeptide has at least 50%, such as at least 75%,at least 90%, at least 95% or at least 100% of the lysozyme activity ofSEQ ID NO: 45. In one embodiment, lysozyme activity is determined asdescribed in example 1.

In one embodiment, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 44. In one embodiment, the polypeptidepreferably comprises or consists of the amino acid sequence of SEQ IDNO: 45; comprises the amino acid sequence of SEQ ID NO: 45 and aN-terminal and/or C-terminal His-tag and/or HQ-tag; comprises the aminoacid sequence of SEQ ID NO: 45 and a N-terminal and/or C-terminalextension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids; or is a fragment thereof having lysozyme activity andhaving at least 90% such as at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the length of SEQ ID NO: 45. In one embodiment, thepolypeptide comprises or consists of amino acids 1 to 227 of SEQ ID NO:45. In one embodiment, the polypeptide has been isolated.

In a continuation of the eighteenth aspect, the invention relates topolypeptides having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 43 of at least 100%, e.g., or 100%. In a further embodiment, thepolypeptide has been isolated.

In a continuation of the eighteenth aspect, the invention relates tovariants of SEQ ID NO: 45 having lysozyme activity comprising one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 45 is not more than 0, e.g. or 1.In one embodiment, the number of substitutions and/or deletions and/orinsertions in SEQ ID NO: 45 is not more than 0, e.g. or 1. In a furtherembodiment, the number of substitutions, preferably conservativesubstitutions, in SEQ ID NO: 45 is not more than 0, e.g. or 1. Examplesof amino acid changes and conservative substitutions are described inthe fourth aspect of the invention.

In an embodiment of the eighteenth aspect, the variant has at least 50%,such as at least 75%, at least 90%, at least 95% or at least 100% of thelysozyme activity of SEQ ID NO: 45. In one embodiment, lysozyme activityis determined as described in example 1.

Taxonomic and Structural Families

In an embodiment, the LAD catalytic domain comprises one or more motifI: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, theLAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP(SEQ ID NO: 318). In an embodiment, the LED comprises one or more motifIII: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). Inan embodiment, the LAD catalytic domain comprises one or more motif I:AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317) and the LED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or moremotif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:319). In an embodiment, the LAD catalytic domain comprises one or moremotif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or moremotif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises oneor more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ IDNO: 319).

In one embodiment, the polypeptide having lysozyme activity is obtainedor is obtainable from the taxonomic phylum Ascomycota, preferably thetaxonomic subphylum Pezizomycotina and is preferably is selected fromthe group selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ IDNO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39,SEQ ID NO: 42 and SEQ ID NO: 45.

In one embodiment, the polypeptide having lysozyme activity is obtainedor is obtainable from the taxonomic class Eurotiomycetes, preferably thetaxonomic order Eurotiales and is more preferably selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ IDNO: 30 and SEQ ID NO: 36.

In one embodiment, the polypeptide having lysozyme activity is obtainedor is obtainable from the taxonomic order Eurotiales, preferably thetaxonomic family Aspergillaceae and is more preferably selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12, SEQ IDNO: 15, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27 and SEQ ID NO: 30.

In one embodiment, the polypeptide having lysozyme activity is obtainedor is obtainable from the taxonomic order Eurotiales, preferably thetaxonomic family Trichocomaceae and is more preferably selected from thegroup consisting of SEQ ID NO: 9 and SEQ ID NO: 36.

In one embodiment, the polypeptide having lysozyme activity is obtainedor is obtainable from the taxonomic class Sordariomycetes and ispreferably selected from the group selected from SEQ ID NO: 18, SEQ IDNO: 33, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45.

In one embodiment, the polypeptide having lysozyme activity is obtainedor is obtainable from the taxonomic order Sordariales, preferably thetaxonomic family Chaetomiaceae and is more preferably selected from thegroup consisting of SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 39 and SEQID NO: 45.

In one embodiment, the polypeptide having lysozyme activity is obtainedor is obtainable from the taxonomic order Hypocreales, preferably thetaxonomic family Clavicipitaceae and is more preferably selected fromthe group consisting of SEQ ID NO: 42.

Sources of Polypeptides Having Lysozyme Activity

A polypeptide having lysozyme activity of the present invention may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by a polynucleotideis produced by the source or by a strain in which the polynucleotidefrom the source has been inserted. In one aspect, the polypeptideobtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a polypeptide having lysozyme activity from a fungus ofthe class Eurotiomycetes, such as from the order Eurotiales, or from thefamily Aspergillaceae, or from the genus Penicillium, or from thespecies Penicillium simplicissimum, Penicillium vasconiae, Penicilliumantarcticum, Penicillium wellingtonense, Penicillium roseopurpureum orPenicillium virgatum.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a polypeptide having lysozyme activity from a fungus ofthe class Eurotiomycetes, such as from the order Eurotiales, or from thefamily Aspergillaceae, or from the genus Aspergillus, or from thespecies Aspergillus sp. XZ2668 or Aspergillus niveus.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a polypeptide having lysozyme activity from a fungus ofthe class Eurotiomycetes, such as from the order Eurotiales, or from thefamily Trichocomaceae, or from the genus Talaromyces, or from thespecies Talaromyces proteolyticus or Talaromyces atricola.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a polypeptide having lysozyme activity from a fungus ofthe class Sordariomycetes, such as from the order Hypocreales, or fromthe family Clavicipitaceae, or from the genus Metarhizium, or from thespecies Metarhizium carneum.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a polypeptide having lysozyme activity from a fungus ofthe class Sordariomycetes, such as from the order Sordariales, or fromthe family Chaetomiaceae, or from the genus Ovatospora, or from thespecies Ovatospora brasiliensis.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a polypeptide having lysozyme activity from a fungus ofthe class Sordariomycetes, such as from the order Sordariales, or fromthe family Chaetomiaceae, or from the genus Chaetomium, or from thespecies Chaetomium sp. ZY369.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a polypeptide having lysozyme activity from a fungus ofthe class Sordariomycetes, such as from the order Sordariales, or fromthe family Chaetomiaceae, or from the genus Trichocladium, or from thespecies Trichocladium asperum.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a polypeptide having lysozyme activity from a fungus ofthe class Sordariomycetes, such as from the order Sordariales, or fromthe family Chaetomiaceae, or from the genus Thielavia, or from thespecies Thielavia terrestris.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to polynucleotides encoding apolypeptide of the present invention, as described herein. In anembodiment, the polynucleotide encoding the polypeptide of the presentinvention has been isolated.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well-known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Trichophaea or a strainof Trichoderma, or a related organism and thus, for example, may be anallelic or species variant of the polypeptide encoding region of thepolynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

In some embodiments, the polypeptide is heterologous to the recombinanthost cell.

In some embodiments, at least one of the one or more control sequencesis heterologous to the polynucleotide encoding the polypeptide.

In some embodiments, the recombinant host cell comprises at least twocopies, e.g., three, four, or five, of the polynucleotide of the presentinvention.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia MicrobioL (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. MicrobioL Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. MicrobioL 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. MicrobioL 65: 3800-3804), or conjugation (see,e.g., Clewell, 1981, MicrobioL Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally, (b) recovering thepolypeptide.

In one aspect, the cell is a Penicillium simplicissimum cell. In oneaspect, the cell is a Penicillium vasconiae cell. In one aspect, thecell is a Talaromyces proteolyticus cell. In one aspect, the cell is anAspergillus sp. XZ2668 cell. In one aspect, the cell is a Penicilliumantarcticum cell. In one aspect, the cell is a Ovatospora brasiliensiscell. In one aspect, the cell is a Penicillium wellingtonense cell. Inone aspect, the cell is a Penicillium roseopurpureum cell. In oneaspect, the cell is a Penicillium virgatum cell. In one aspect, the cellis an Aspergillus niveus cell. In one aspect, the cell is a Chaetomiumsp. ZY369 cell. In one aspect, the cell is a Talaromyces atricola cell.In one aspect, the cell is a Trichocladium asperum cell. In one aspect,the cell is a Metarhizium carneum cell. In one aspect, the cell is aThielavia terrestris cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally, (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the fermentation mediumby conventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.

Plant cells and specific plant cell compartments, such as chloroplasts,apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are alsoconsidered to be a plant part.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. In some embodiments, the composition is a cell-killed wholebroth containing organic acid(s), killed cells and/or cell debris, andculture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In some embodiments, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In some embodiments, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In someembodiments, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis. In some embodiments, the cell-killed whole broth orcomposition contains the spent cell culture medium, extracellularenzymes, and killed filamentous fungal cells. In some embodiments, themicrobial cells present in the cell-killed whole broth or compositioncan be permeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in the polypeptide of the invention. The term “enriched”indicates that the lysozyme activity of the composition has beenincreased, e.g., with an enrichment factor of at least 1.1, such as atleast 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, atleast 3.0, at least 4.0, at least 5.0, at least 10.

In a preferred embodiment, the composition comprises one or more LYSpolypeptides having lysozyme activity selected from the list consistingof SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO:15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ IDNO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 andSEQ ID NO: 45.

In an embodiment, the composition comprises the polypeptide of theinvention and one or more formulating agents, as described below.

The compositions may further comprise multiple enzymatic activities,such as one or more (e.g., several) enzymes selected from the groupconsisting of phytase, xylanase, galactanase, alpha-galactosidase,beta-galactosidase, protease, phospholipase A1, phospholipase A2,lysophospholipase, phospholipase C, phospholipase D, amylase, lysozyme,arabinofuranosidase, beta-xylosidase, acetyl xylan esterase, feruloylesterase, cellulase, cellobiohydrolases, beta-glucosidase, pullulanase,and beta-glucanase or any combination thereof.

The compositions may further comprise one or more probiotics. In anembodiment, the probiotic is selected from the group consisting ofBacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens,Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillusmegaterium, Bacillus coagulans, Bacillus circulans, Bifidobacteriumbifidum, Bifidobacterium animalis, Bifidobacterium sp., Carnobacteriumsp., Clostridium butyricum, Clostridium sp., Enterococcus faecium,Enterococcus sp., Lactobacillus sp., Lactobacillus acidophilus,Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillusreuteri, Lactobacillus salivarius, Lactococcus Lactis, Lactococcus sp.,Leuconostoc sp., Megasphaera elsdenii, Megasphaera sp., Pediococsusacidilactici, Pediococcus sp., Propionibacterium thoenii,Propionibacterium sp. and Streptococcus sp. or any combination thereof.

In an embodiment, the composition comprises one or more formulatingagents as disclosed herein, preferably one or more of the compoundsselected from the list consisting of glycerol, ethylene glycol, 1,2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodiumbenzoate, potassium sorbate, sodium sulfate, potassium sulfate,magnesium sulfate, sodium thiosulfate, calcium carbonate, sodiumcitrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin,maltodextrin, cyclodextrin, wheat, PVA, acetate, phosphate, kaolin andcellulose.

In an embodiment, the composition comprises one or more componentsselected from the list consisting of vitamins, minerals and amino acids.

Formulation

The enzyme of the invention may be formulated as a liquid or a solid.For a liquid formulation, the formulating agent may comprise a polyol(such as e.g. glycerol, ethylene glycol or propylene glycol), a salt(such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or asugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, andsorbitol). Thus in one embodiment, the composition is a liquidcomposition comprising the polypeptide of the invention and one or moreformulating agents selected from the list consisting of glycerol,ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, sodiumchloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose,and sorbitol. The liquid formulation may be sprayed onto the feed afterit has been pelleted or may be added to drinking water given to theanimals.

For a solid formulation, the formulation may be for example as agranule, spray dried powder or agglomerate (e.g. as disclosed inWO2000/70034). The formulating agent may comprise a salt (organic orinorganic zinc, sodium, potassium or calcium salts such as e.g. such ascalcium acetate, calcium benzoate, calcium carbonate, calcium chloride,calcium citrate, calcium sorbate, calcium sulfate, potassium acetate,potassium benzoate, potassium carbonate, potassium chloride, potassiumcitrate, potassium sorbate, potassium sulfate, sodium acetate, sodiumbenzoate, sodium carbonate, sodium chloride, sodium citrate, sodiumsulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride,zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugarderivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol).

In one embodiment, the composition is a solid composition, such as aspray dried composition, comprising the LYS polypeptide of the inventionand one or more formulating agents selected from the list consisting ofsodium chloride, sodium benzoate, potassium sorbate, sodium sulfate,potassium sulfate, magnesium sulfate, sodium thiosulfate, calciumcarbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose,starch, kaolin, maltodextrin, cyclodextrin, wheat, PVA, acetate,phosphate and cellulose. In a preferred embodiment, the formulatingagent is selected from one or more of the following compounds: sodiumsulfate, dextrin, cellulose, sodium thiosulfate, magnesium sulfate andcalcium carbonate.

The present invention also relates to enzyme granules/particlescomprising the LYS polypeptide of the invention optionally combined withone or more additional enzymes. The granule is composed of a core, andoptionally one or more coatings (outer layers) surrounding the core.

Typically the granule/particle size, measured as equivalent sphericaldiameter (volume based average particle size), of the granule is 20-2000μm, particularly 50-1500 μm, 100-1500 μm or 250-1200 μm.

The core can be prepared by granulating a blend of the ingredients,e.g., by a method comprising granulation techniques such ascrystallization, precipitation, pan-coating, fluid bed coating, fluidbed agglomeration, rotary atomization, extrusion, prilling,spheronization, size reduction methods, drum granulation, and/or highshear granulation.

Methods for preparing the core can be found in Handbook of PowderTechnology; Particle size enlargement by C. E. Capes; Volume 1; 1980;Elsevier. Preparation methods include known feed and granule formulationtechnologies, e.g.:

a) spray dried products, wherein a liquid enzyme-containing solution isatomized in a spray drying tower to form small droplets which duringtheir way down the drying tower dry to form an enzyme-containingparticulate material;

b) layered products, wherein the enzyme is coated as a layer around apre-formed inert core particle, wherein an enzyme-containing solution isatomized, typically in a fluid bed apparatus wherein the pre-formed coreparticles are fluidized, and the enzyme-containing solution adheres tothe core particles and dries up to leave a layer of dry enzyme on thesurface of the core particle. Particles of a desired size can beobtained this way if a useful core particle of the desired size can befound. This type of product is described in, e.g., WO 97/23606;

c) absorbed core particles, wherein rather than coating the enzyme as alayer around the core, the enzyme is absorbed onto and/or into thesurface of the core. Such a process is described in WO 97/39116.

d) extrusion or pelletized products, wherein an enzyme-containing pasteis pressed to pellets or under pressure is extruded through a smallopening and cut into particles which are subsequently dried. Suchparticles usually have a considerable size because of the material inwhich the extrusion opening is made (usually a plate with bore holes)sets a limit on the allowable pressure drop over the extrusion opening.Also, very high extrusion pressures when using a small opening increaseheat generation in the enzyme paste, which is harmful to the enzyme;

e) prilled products, wherein an enzyme-containing powder is suspended inmolten wax and the suspension is sprayed, e.g., through a rotating diskatomiser, into a cooling chamber where the droplets quickly solidify(Michael S. Showell (editor); Powdered detergents; Surfactant ScienceSeries; 1998; vol. 71; page 140-142; Marcel Dekker). The productobtained is one wherein the enzyme is uniformly distributed throughoutan inert material instead of being concentrated on its surface. AlsoU.S. Pat. Nos. 4,016,040 and 4,713,245 are documents relating to thistechnique;

f) mixer granulation products, wherein a liquid is added to a dry powdercomposition of, e.g., conventional granulating components, the enzymebeing introduced either via the liquid or the powder or both. The liquidand the powder are mixed and as the moisture of the liquid is absorbedin the dry powder, the components of the dry powder will start to adhereand agglomerate and particles will build up, forming granulatescomprising the enzyme. Such a process is described in U.S. Pat. No.4,106,991 and related documents EP 170360, EP 304332, EP 304331, WO90/09440 and WO 90/09428. In a particular product of this processwherein various high-shear mixers can be used as granulators, granulatesconsisting of enzyme as enzyme, fillers and binders etc. are mixed withcellulose fibres to reinforce the particles to give the so-calledT-granulate. Reinforced particles, being more robust, release lessenzymatic dust.

g) size reduction, wherein the cores are produced by milling or crushingof larger particles, pellets, tablets, briquettes etc. containing theenzyme. The wanted core particle fraction is obtained by sieving themilled or crushed product. Over and undersized particles can berecycled. Size reduction is described in (Martin Rhodes (editor);Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons);

h) fluid bed granulation, which involves suspending particulates in anair stream and spraying a liquid onto the fluidized particles vianozzles. Particles hit by spray droplets get wetted and become tacky.The tacky particles collide with other particles and adhere to them andform a granule;

i) the cores may be subjected to drying, such as in a fluid bed drier.Other known methods for drying granules in the feed or detergentindustry can be used by the skilled person. The drying preferably takesplace at a product temperature of from 25 to 90° C. For some enzymes itis important the cores comprising the enzyme contain a low amount ofwater before coating. If water sensitive enzymes are coated beforeexcessive water is removed, it will be trapped within the core and itmay affect the activity of the enzyme negatively. After drying, thecores preferably contain 0.1-10% w/w water.

The core may include additional materials such as fillers, fibrematerials (cellulose or synthetic fibres), stabilizing agents,solubilizing agents, suspension agents, viscosity regulating agents,light spheres, plasticizers, salts, lubricants and fragrances.

The core may include a binder, such as synthetic polymer, wax, fat, orcarbohydrate.

The core may include a salt of a multivalent cation, a reducing agent,an antioxidant, a peroxide decomposing catalyst and/or an acidic buffercomponent, typically as a homogenous blend.

In one embodiment, the core comprises a material selected from the groupconsisting of salts (such as calcium acetate, calcium benzoate, calciumcarbonate, calcium chloride, calcium citrate, calcium sorbate, calciumsulfate, potassium acetate, potassium benzoate, potassium carbonate,potassium chloride, potassium citrate, potassium sorbate, potassiumsulfate, sodium acetate, sodium benzoate, sodium carbonate, sodiumchloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate,zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zincsulfate), starch or a sugar or sugar derivative (such as e.g. sucrose,dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such ase.g. sucrose, dextrin, glucose, lactose, sorbitol), small organicmolecules, starch, flour, cellulose and minerals and clay minerals (alsoknown as hydrous aluminium phyllosilicates). In one embodiment, the corecomprises a clay mineral such as kaolinite or kaolin.

The core may include an inert particle with the enzyme absorbed into it,or applied onto the surface, e.g., by fluid bed coating.

The core may have a diameter of 20-2000 μm, particularly 50-1500 μm,100-1500 μm or 250-1200 μm.

The core may be surrounded by at least one coating, e.g., to improve thestorage stability, to reduce dust formation during handling, or forcoloring the granule. The optional coating(s) may include a salt and/orwax and/or flour coating, or other suitable coating materials.

The coating may be applied in an amount of at least 0.1% by weight ofthe core, e.g., at least 0.5%, 1% or 5%. The amount may be at most 100%,70%, 50%, 40% or 30%.

The coating is preferably at least 0.1 μm thick, particularly at least0.5 μm, at least 1 μm or at least 5 μm. In some embodiments thethickness of the coating is below 100 μm, such as below 60 μm, or below40 μm.

The coating should encapsulate the core unit by forming a substantiallycontinuous layer. A substantially continuous layer is to be understoodas a coating having few or no holes, so that the core unit isencapsulated or enclosed with few or no uncoated areas. The layer orcoating should in particular be homogeneous in thickness.

The coating can further contain other materials as known in the art,e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/orbinders, such as titanium dioxide, kaolin, calcium carbonate or talc.

A salt coating may comprise at least 60% by weight of a salt, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% by weight.

The salt may be added from a salt solution where the salt is completelydissolved or from a salt suspension wherein the fine particles are lessthan 50 μm, such as less than 10 μm or less than 5 μm.

The salt coating may comprise a single salt or a mixture of two or moresalts. The salt may be water soluble, in particular having a solubilityat least 0.1 g in 100 g of water at 20° C., preferably at least 0.5 gper 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 gper 100 g water.

The salt may be an inorganic salt, e.g., salts of sulfate, sulfite,phosphate, phosphonate, nitrate, chloride or carbonate or salts ofsimple organic acids (less than 10 carbon atoms, e.g., 6 or less carbonatoms) such as citrate, malonate or acetate. Examples of cations inthese salts are alkali or earth alkali metal ions, the ammonium ion ormetal ions of the first transition series, such as sodium, potassium,magnesium, calcium, zinc or aluminium. Examples of anions includechloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate,phosphate, monobasic phosphate, dibasic phosphate, hypophosphite,dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate,metasilicate, citrate, malate, maleate, malonate, succinate, sorbate,lactate, formate, acetate, butyrate, propionate, benzoate, tartrate,ascorbate or gluconate. In particular alkali- or earth alkali metalsalts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride orcarbonate or salts of simple organic acids such as citrate, malonate oracetate may be used.

The salt in the coating may have a constant humidity at 20° C. above60%, particularly above 70%, above 80% or above 85%, or it may beanother hydrate form of such a salt (e.g., anhydrate). The salt coatingmay be as described in WO1997/05245, WO1998/54980, WO1998/55599,WO2000/70034, WO2006/034710, WO2008/017661, WO2008/017659,WO2000/020569, WO2001/004279, WO1997/05245, WO2000/01793, WO2003/059086,WO2003/059087, WO2007/031483, WO2007/031485, WO2007/044968,WO2013/192043, WO2014/014647 and WO2015/197719 or polymer coating suchas described in WO 2001/00042.

Specific examples of suitable salts are NaCl (CH20° C.=76%), Na2CO3(CH20° C.=92%), NaNO3 (CH20° C.=73%), Na2HPO4 (CH20° C.=95%), Na3PO4(CH25° C.=92%), NH4Cl (CH20° C.=79.5%), (NH4)2HPO4 (CH20° C.=93.0%),NH4H2PO4 (CH20° C.=93.1%), (NH4)2504 (CH20° C.=81.1%), KCl (CH20°C.=85%), K2HPO4 (CH20° C.=92%), KH2PO4 (CH20° C.=96.5%), KNO3 (CH20°C.=93.5%), Na2SO4 (CH20° C.=93%), K2504 (CH20° C.=98%), KHSO4 (CH20°C.=86%), MgSO4 (CH20° C.=90%), ZnSO4 (CH20° C.=90%) and sodium citrate(CH25° C.=86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4,Mg(NO3)2, magnesium acetate, calcium acetate, calcium benzoate, calciumcarbonate, calcium chloride, calcium citrate, calcium sorbate, calciumsulfate, potassium acetate, potassium benzoate, potassium carbonate,potassium chloride, potassium citrate, potassium sorbate, sodiumacetate, sodium benzoate, sodium citrate, sodium sulfate, zinc acetate,zinc benzoate, zinc carbonate, zinc chloride, zinc citrate and zincsorbate.

The salt may be in anhydrous form, or it may be a hydrated salt, i.e. acrystalline salt hydrate with bound water(s) of crystallization, such asdescribed in WO 99/32595. Specific examples include anhydrous sodiumsulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfateheptahydrate (MgSO4.7H2O), zinc sulfate heptahydrate (ZnSO4.7H2O),sodium phosphate dibasic heptahydrate (Na2HPO4.7H2O), magnesium nitratehexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesiumacetate tetrahydrate.

Preferably the salt is applied as a solution of the salt, e.g., using afluid bed.

A wax coating may comprise at least 60% by weight of a wax, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% by weight.

Specific examples of waxes are polyethylene glycols; polypropylenes;Carnauba wax; Candelilla wax; bees wax; hydrogenated plant oil or animaltallow such as polyethylene glycol (PEG), methyl hydroxy-propylcellulose (MHPC), polyvinyl alcohol (PVA), hydrogenated ox tallow,hydrogenated palm oil, hydrogenated cotton seeds and/or hydrogenated soybean oil; fatty acid alcohols; mono-glycerides and/or di-glycerides,such as glyceryl stearate, wherein stearate is a mixture of stearic andpalmitic acid; micro-crystalline wax; paraffin's; and fatty acids, suchas hydrogenated linear long chained fatty acids and derivatives thereof.A preferred wax is palm oil or hydrogenated palm oil.

The granule may comprise a core comprising the LYS polypeptide of theinvention, one or more salt coatings and one or more wax coatings.Examples of enzyme granules with multiple coatings are shown inWO1993/07263, WO1997/23606 and WO2016/149636.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. The coating materials can be waxy coating materialsand film-forming coating materials. Examples of waxy coating materialsare poly(ethylene oxide) products (polyethyleneglycol, PEG) with meanmolar weights of 1000 to 20000; ethoxylated nonylphenols having from 16to 50 ethylene oxide units; ethoxylated fatty alcohols in which thealcohol contains from 12 to 20 carbon atoms and in which there are 15 to80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di-and triglycerides of fatty acids. Examples of film-forming coatingmaterials suitable for application by fluid bed techniques are given inGB 1483591.

The granulate may further comprise one or more additional enzymes. Eachenzyme will then be present in more granules securing a more uniformdistribution of the enzymes, and also reduces the physical segregationof different enzymes due to different particle sizes. Methods forproducing multi-enzyme co-granulates is disclosed in the ip.comdisclosure IPCOM000200739D.

Another example of formulation of enzymes by the use of co-granulates isdisclosed in WO 2013/188331.

The present invention also relates to protected enzymes preparedaccording to the method disclosed in EP 238,216.

Thus, in a further aspect, the present invention provides a granule,which comprises:

(a) a core comprising a LYS polypeptide having lysozyme activityaccording to the invention, and

(b) a coating consisting of one or more layer(s) surrounding the core.

In one embodiment, the coating comprises a salt coating as describedherein. In one embodiment, the coating comprises a wax coating asdescribed herein. In one embodiment, the coating comprises a saltcoating followed by a wax coating as described herein.

Animal Feed Additives

The present invention also relates to animal feed additives comprisingone or more LYS polypeptides having lysozyme activity. Thus, in oneembodiment, the invention relates to an animal feed additive comprisinga LYS polypeptide, wherein:

-   -   (a) the polypeptide has lysozyme activity;    -   (b) the polypeptide comprises one or more LAD catalytic domains;        and    -   (c) the LAD catalytic domain gives a domT score of at least 170        when queried using a Profile Hidden Markov Model prepared using        SEQ ID NOs: 46 to 187 and hmmbuild software program, and wherein        the query is carried out using hmmscan software program by the        Method of Determining the Lysozyme Enhancing Domain by HMM.

In an embodiment, the polypeptide further comprises one or more lysozymeenhancing domains, wherein the lysozyme enhancing domain gives a domTscore of at least 100 when queried using a Profile Hidden Markov Modelprepared using SEQ ID NOs: 188 to 316 and hmmbuild software program, andwherein the query is carried out using the hmmscan software program.

In an embodiment, the domT score of the LAD catalytic domain is at least175, preferably at least 180, more preferably at least 185, even morepreferably at least 190, even more preferably at least 195, or mostpreferably at least 200. In an embodiment, the domT score of the LED isat least 103, preferably at least 106, more preferably at least 109,more preferably at least 112, more preferably at least 115, morepreferably at least 118, even more preferably at least 121, or mostpreferably at least 124. Preferred combinations of domT scores are asdisclosed in the first aspect of the invention.

In an embodiment, the LAD catalytic domain comprises one or more motifI: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, theLAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP(SEQ ID NO: 318). In an embodiment, the LED comprises one or more motifIII: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). Inan embodiment, the LAD catalytic domain comprises one or more motif I:AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317) and the LED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or moremotif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:319). In an embodiment, the LAD catalytic domain comprises one or moremotif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or moremotif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises oneor more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ IDNO: 319).

In another aspect, the invention relates to animal feed additivescomprising one or more LYS polypeptides having lysozyme activity,wherein the polypeptide is selected from the group consisting of:

-   -   (a) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 3;    -   (b) a polypeptide having at least 84%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 27;    -   (j) a polypeptide having at least 84%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 36;    -   (m) a polypeptide having at least 84%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 42;    -   (o) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 45;    -   (p) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID        NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO:        24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36,        SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45        positions;    -   (q) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o)        or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;    -   (r) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o)        or (p) and a N-terminal and/or C-terminal extension of up to 10        amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;        and    -   (s) a fragment of the polypeptide of (a), (b), (c), (d), (e),        (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having        lysozyme activity and having at least 90% of the length of the        mature polypeptide.

In an embodiment, the LAD catalytic domain comprises one or more motifI: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, theLAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP(SEQ ID NO: 318). In an embodiment, the LED comprises one or more motifIII: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). Inan embodiment, the LAD catalytic domain comprises one or more motif I:AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317) and the LED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or moremotif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:319). In an embodiment, the LAD catalytic domain comprises one or moremotif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or moremotif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises oneor more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ IDNO: 319).

In one embodiment, the polypeptide is of fungal origin. In anembodiment, the polypeptide is obtained or obtainable from the taxonomicphylum Ascomycota, preferably the taxonomic subphylum Pezizomycotina.

In an embodiment, the amount of enzyme in the animal feed additive isbetween 0.001% and 10% by weight of the composition.

In an embodiment, the animal feed additive comprises one or moreformulating agents, preferably as described herein above.

In an embodiment, the animal feed additive comprises one or moreadditional enzymes, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or moreprobiotics, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or morevitamins, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or moreminerals, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or more aminoacids, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or moreprebiotics, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or more organicacids, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or morephytogenics, preferably as described herein below.

Animal Feed

The present invention also relates to animal feed compositionscomprising one or more lysozymes of the invention. In one embodiment,the invention relates to an animal feed comprising the granule asdescribed herein and plant based material. In one embodiment, theinvention relates to an animal feed comprising the animal feed additiveas described herein and plant based material.

Animal feed compositions or diets have a relatively high content ofprotein. Poultry and pig diets can be characterised as indicated inTable B of WO 01/58275, columns 2-3. Fish diets can be characterised asindicated in column 4 of this Table B. Furthermore such fish dietsusually have a crude fat content of 200-310 g/kg.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least onepolypeptide having lysozyme activity as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e. Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen by, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein as defined above.

The animal feed composition of the invention may also contain animalprotein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal,typically in an amount of 0-25%. The animal feed composition of theinvention may also comprise Dried Distillers Grains with Solubles(DDGS), typically in amounts of 0-30%.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

The animal feed may comprise vegetable proteins. In particularembodiments, the protein content of the vegetable proteins is at least10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may bederived from vegetable protein sources, such as legumes and cereals, forexample, materials from plants of the families Fabaceae (Leguminosae),Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupinmeal, rapeseed meal, and combinations thereof.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g., soybean, lupine,pea, or bean. In another particular embodiment, the vegetable proteinsource is material from one or more plants of the family Chenopodiaceae,e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetableprotein sources are rapeseed, and cabbage. In another particularembodiment, soybean is a preferred vegetable protein source. Otherexamples of vegetable protein sources are cereals such as barley, wheat,rye, oat, maize (corn), rice, and sorghum.

Animal diets can e.g. be manufactured as mash feed (non-pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Enzymes canbe added as solid or liquid enzyme formulations. For example, for mashfeed a solid or liquid enzyme formulation may be added before or duringthe ingredient mixing step. For pelleted feed the (liquid or solid)lysozyme/enzyme preparation may also be added before or during the feedingredient step. Typically a liquid lysozyme/enzyme preparationcomprises the polypeptide having lysozyme activity of the inventionoptionally with a polyol, such as glycerol, ethylene glycol or propyleneglycol, and is added after the pelleting step, such as by spraying theliquid formulation onto the pellets. The enzyme may also be incorporatedin a feed additive or premix.

Alternatively, the polypeptide having lysozyme activity can be preparedby freezing a mixture of liquid enzyme solution with a bulking agentsuch as ground soybean meal, and then lyophilizing the mixture.

The final enzyme concentration in the diet is within the range of0.01-200 mg enzyme protein per kg diet, preferably between 0.05-100mg/kg diet, more preferably 0.1-50 mg, even more preferably 0.2-20 mgenzyme protein per kg animal diet.

It is at present contemplated that the enzyme is administered in one ormore of the following amounts (dosage ranges): 0.01-200; 0.05-100;0.1-50; 0.2-20; 0.1-1; 0.2-2; 0.5-5; or 1-10; —all these ranges being inmg LYS polypeptide protein per kg feed (ppm).

For determining mg LYS polypeptide protein per kg feed, the LYSpolypeptide is purified from the feed composition, and the specificactivity of the purified LYS polypeptide is determined using a relevantassay (see under lysozyme activity). The lysozyme activity of the feedcomposition as such is also determined using the same assay, and on thebasis of these two determinations, the dosage in mg lysozyme protein perkg feed is calculated.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The same principles apply for determining mg LYS polypeptide protein infeed additives. Of course, if a sample is available of the LYSpolypeptide used for preparing the feed additive or the feed, thespecific activity is determined from this sample (no need to purify theLYS polypeptide from the feed composition or the additive).

Thus in a further aspect, the present invention also relates to ananimal feed comprising one or more LYS polypeptides having lysozymeactivity and plant based material. In another aspect, the presentinvention also relates to an animal feed comprising the animal feedadditive of the invention (as described herein above) and plant basedmaterial.

In one embodiment, the invention relates to an animal feed comprisingplant based material and a LYS polypeptide, wherein the polypeptide (a)has lysozyme activity and (b) comprises one or more LAD catalyticdomains; wherein the LAD catalytic domain gives a domT score of at least180 when queried using a Profile Hidden Markov Model (HMM) preparedusing SEQ ID NOs: 46 to 187 and hmmbuild software program, and whereinthe query is carried out using hmmscan software program by the Method ofDetermining the LAD Catalytic Domain by HMM.

In an embodiment, the polypeptide further comprises one or more lysozymeenhancing domains, wherein the lysozyme enhancing domain gives a domTscore of at least 100 when queried using a Profile Hidden Markov Modelprepared using SEQ ID NOs: 188 to 316 and hmmbuild software program, andwherein the query is carried out using the hmmscan software program bythe Method of Determining the Lysozyme.

In an embodiment, the domT score of the LAD catalytic domain is at least175, preferably at least 180, more preferably at least 185, even morepreferably at least 190, even more preferably at least 195, or mostpreferably at least 200. In an embodiment, the domT score of the LED isat least 103, preferably at least 106, more preferably at least 109,more preferably at least 112, more preferably at least 115, morepreferably at least 118, even more preferably at least 121, or mostpreferably at least 124. Preferred combinations of domT scores are asdisclosed in the first aspect of the invention.

In an embodiment, the LAD catalytic domain comprises one or more motifI: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, theLAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP(SEQ ID NO: 318). In an embodiment, the LED comprises one or more motifIII: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). Inan embodiment, the LAD catalytic domain comprises one or more motif I:AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317) and the LED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or moremotif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:319). In an embodiment, the LAD catalytic domain comprises one or moremotif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or moremotif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises oneor more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ IDNO: 319).

In another aspect, the invention relates to an animal feed comprisingplant based material and one or more LYS polypeptides having lysozymeactivity, wherein the polypeptide is selected from the group consistingof:

-   -   (a) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 3;    -   (b) a polypeptide having at least 84%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 27;    -   (j) a polypeptide having at least 84%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 36;    -   (m) a polypeptide having at least 84%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 42;    -   (o) a polypeptide having at least 80%, such as at least 85%, at        least 90% or at least 95% sequence identity to the polypeptide        of SEQ ID NO: 45;    -   (p) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID        NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO:        24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36,        SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45        positions;    -   (q) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o)        or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;    -   (r) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o)        or (p) and a N-terminal and/or C-terminal extension of up to 10        amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;        and    -   (s) a fragment of the polypeptide of (a), (b), (c), (d), (e),        (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having        lysozyme activity and having at least 90% of the length of the        mature polypeptide.

In an embodiment, the LAD catalytic domain comprises one or more motifI: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317). In an embodiment, theLAD catalytic domain comprises one or more motif II V[G/A]XLCQXVQXSAYP(SEQ ID NO: 318). In an embodiment, the LED comprises one or more motifIII: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). Inan embodiment, the LAD catalytic domain comprises one or more motif I:AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318). In an embodiment, the LAD catalyticdomain comprises one or more motif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ IDNO: 317) and the LED comprises one or more motif III:[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO: 319). In anembodiment, the LAD catalytic domain comprises one or more motif IIV[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises one or moremotif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:319). In an embodiment, the LAD catalytic domain comprises one or moremotif I: AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and one or moremotif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318) and the LED comprises oneor more motif III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ IDNO: 319).

In one embodiment, the polypeptide is of fungal origin. In anembodiment, the polypeptide is obtained or obtainable from the taxonomicphylum Ascomycota, preferably the taxonomic subphylum Pezizomycotina.

In an embodiment, the plant based material is selected from the groupconsisting of legumes, cereals, oats, rye, barley, wheat, maize, corn,sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean,wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjimbean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil,peanut, Spanish peanut, canola, rapeseed (oilseed rape), rice, beet,cabbage, sugar beet, spinach, quinoa, or pea, in a processed formthereof (such as soybean meal, rapeseed meal) or any combinationthereof.

In a further embodiment, the animal feed has been pelleted.

Additional Enzymes

In another embodiment, the compositions described herein optionallyinclude one or more enzymes. Enzymes can be classified on the basis ofthe handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also theENZYME site at the internet: http://www.expasy.ch/enzyme/. ENZYME is arepository of information relative to the nomenclature of enzymes. It isprimarily based on the recommendations of the Nomenclature Committee ofthe International Union of Biochemistry and Molecular Biology (IUB-MB),Academic Press, Inc., 1992, and it describes each type of characterizedenzyme for which an EC (Enzyme Commission) number has been provided(Bairoch A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305).This IUB-MB Enzyme nomenclature is based on their substrate specificityand occasionally on their molecular mechanism; such a classificationdoes not reflect the structural features of these enzymes.

Another classification of certain glycoside hydrolase enzymes, such asendoglucanase, xylanase, galactanase, mannanase, dextranase, lysozymeand galactosidase is described in Henrissat et al, “Thecarbohydrate-active enzymes database (CAZy) in 2013”, Nucl. Acids Res.(1 Jan. 2014) 42 (D1): D490-D495; see also www.cazy.org.

Thus the composition of the invention may also comprise at least oneother enzyme selected from the group comprising of phytase (EC 3.1.3.8or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89);alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1(EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4);amylase such as, for example, alpha-amylase (EC 3.2.1.1);arabinofuranosidase (EC 3.2.1.55); beta-xylosidase (EC 3.2.1.37); acetylxylan esterase (EC 3.1.1.72); feruloyl esterase (EC 3.1.1.73); cellulase(EC 3.2.1.4); cellobiohydrolases (EC 3.2.1.91); beta-glucosidase (EC3.2.1.21); pullulanase (EC 3.2.1.41), alpha-mannosidase (EC 3.2.1.24),mannanase (EC 3.2.1.25) and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6),or any mixture thereof.

In a particular embodiment, the composition of the invention comprises aphytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially availablephytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P, Ronozyme®NP and Ronozyme® HiPhos (DSM Nutritional Products), Natuphos™ (BASF),Natuphos™ E (BASF), Finase® and Quantum® Blue (AB Enzymes), OptiPhos®(Huvepharma), AveMix® Phytase (Aveve Biochem), Phyzyme® XP(Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytasesinclude those described in e.g. WO 98/28408, WO 00/43503, and WO03/066847.

In a particular embodiment, the composition of the invention comprises axylanase (EC 3.2.1.8). Examples of commercially available xylanasesinclude Ronozyme® WX (DSM Nutritional Products), Econase® XT and Barley(AB Vista), Xylathin® (Verenium), Hostazym® X (Huvepharma), Axtra® XB(Xylanase/beta-glucanase, DuPont) and Axtra® XAP(Xylanase/amylase/protease, DuPont), AveMix® XG 10 (xylanase/glucanase)and AveMix® 02 CS (xylanase/glucanase/pectinase, Aveve Biochem), andNaturgrain (BASF).

In a particular embodiment, the composition of the invention comprises aprotease (EC 3.4). Examples of commercially available proteases includeRonozyme® ProAct (DSM Nutritional Products).

In a particular embodiment, the composition of the invention comprisesan alpha-amylase (EC 3.2.1.1). Examples of commercially availablealpha-amylases include Ronozyme® A and RONOZYME® RumiStar™ (DSMNutritional Products).

In one embodiment, the composition of the invention comprises amulticomponent enzyme product, such as FRA® Octazyme (Framelco),Ronozyme® G2, Ronozyme® VP and Ronozyme® MultiGrain (DSM NutritionalProducts), Rovabio® Excel or Rovabio® Advance (Adisseo).

Eubiotics

Eubiotics are compounds which are designed to give a healthy balance ofthe micro-flora in the gastrointestinal tract. Eubiotics cover a numberof different feed additives, such as probiotics, prebiotics, phytogenics(essential oils) and organic acids which are described in more detailbelow.

Probiotics

In an embodiment, the animal feed composition further comprises one ormore additional probiotic. In a particular embodiment, the animal feedcomposition further comprises a bacterium from one or more of thefollowing genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus,Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or anycombination thereof.

In a preferred embodiment, animal feed composition further comprises abacterium from one or more of the following strains: Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus,Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, Enterococcus faecium, Enterococcus spp,and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp,Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis,Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillusfarciminus, Lactobacillus rhamnosus, Clostridium butyricum,Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri,Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii,Propionibacteria sp.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus subtilis: 3A-P4 (PTA-6506), 15A-P4(PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01(NRRL-B-50104), BS27 (NRRL B-501 05), BS 18 (NRRL B-50633), BS 278 (NRRLB-50634), DSM 29870, DSM 29871, DSM 32315, NRRL B-50136, NRRL B-50605,NRRL B-50606, NRRL B-50622 and PTA-7547.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus pumilus: NRRL B-50016, ATCC 700385, NRRLB-50885 or NRRL B-50886.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus lichenformis: NRRL B 50015, NRRL B-50621or NRRL B-50623.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus amyloliquefaciens: DSM 29869, DSM 29869,NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B-50606, NRRLB-50013, NRRL B-50151, NRRL B-50141, NRRL B-50147 or NRRL B-50888.

The bacterial count of each of the bacterial strains in the animal feedcomposition is between 1×10⁴ and 1×10¹⁴ CFU/kg of dry matter, preferablybetween 1×10⁶ and 1×10¹² CFU/kg of dry matter, and more preferablybetween 1×10⁷ and 1×10¹¹ CFU/kg of dry matter. In a more preferredembodiment the bacterial count of each of the bacterial strains in theanimal feed composition is between 1×10⁸ and 1×10¹⁰CFU/kg of dry matter.

The bacterial count of each of the bacterial strains in the animal feedcomposition is between 1×10⁵ and 1×10¹⁵ CFU/animal/day, preferablybetween 1×10⁷ and 1×10¹³ CFU/animal/day, and more preferably between1×10⁸ and 1×10¹² CFU/animal/day. In a more preferred embodiment thebacterial count of each of the bacterial strains in the animal feedcomposition is between 1×10⁹ and 1×10¹¹ CFU/animal/day. In oneembodiment, the amount of probiotics is 0.001% to 10% by weight of thecomposition.

In another embodiment, the one or more bacterial strains are present inthe form of a stable spore.

Examples of commercial products are Cylactin® (DSM NutritionalProducts), Alterion (Adisseo), Enviva PRO (DuPont Animal Nutrition),Syncra® (mix enzyme+probiotic, DuPont Animal Nutrition), Ecobiol® andFecinor0 (Norel/Evonik) and GutCare® PY1 (Evonik).

Prebiotics

Prebiotics are substances that induce the growth or activity ofmicroorganisms (e.g., bacteria and fungi) that contribute to thewell-being of their host. Prebiotics are typically non-digestible fibercompounds that pass undigested through the upper part of thegastrointestinal tract and stimulate the growth or activity ofadvantageous bacteria that colonize the large bowel by acting assubstrate for them. Normally, prebiotics increase the number or activityof bifidobacteria and lactic acid bacteria in the GI tract.

Yeast derivatives (inactivated whole yeasts or yeast cell walls) canalso be considered as prebiotics. They often comprisemannan-oligosaccharids, yeast beta-glucans or protein contents and arenormally derived from the cell wall of the yeast, Saccharomycescerevisiae.

In one embodiment, the amount of prebiotics is 0.001% to 10% by weightof the composition. Examples of yeast products are Yang® and Agrimos(Lallemand Animal Nutrition).

Phytogenics

Phytogenics are a group of natural growth promoters or non-antibioticgrowth promoters used as feed additives, derived from herbs, spices orother plants. Phytogenics can be single substances prepared fromessential oils/extracts, essential oils/extracts, single plants andmixture of plants (herbal products) or mixture of essentialoils/extracts/plants (specialized products).

Examples of phytogenics are rosemary, sage, oregano, thyme, clove, andlemongrass. Examples of essential oils are thymol, eugenol, meta-cresol,vaniline, salicylate, resorcine, guajacol, gingerol, lavender oil,ionones, irone, eucalyptol, menthol, peppermint oil, alpha-pinene;limonene, anethol, linalool, methyl dihydrojasmonate, carvacrol,propionic acid/propionate, acetic acid/acetate, butyric acid/butyrate,rosemary oil, clove oil, geraniol, terpineol, citronellol, amyl and/orbenzyl salicylate, cinnamaldehyde, plant polyphenol (tannin), turmericand curcuma extract.

In one embodiment, the amount of phytogeneics is 0.001% to 10% by weightof the composition. Examples of commercial products are Crina® (DSMNutritional Products); Cinergy™, Biacid™, ProHacid™ Classic andProHacid™ Advance™ (all Promivi/Cargill) and Envivo EO (DuPont AnimalNutrition).

Organic Acids

Organic acids (C1-C7) are widely distributed in nature as normalconstituents of plants or animal tissues. They are also formed throughmicrobial fermentation of carbohydrates mainly in the large intestine.They are often used in swine and poultry production as a replacement ofantibiotic growth promoters since they have a preventive effect on theintestinal problems like necrotic enteritis in chickens and Escherichiacoli infection in young pigs. Organic acids can be sold as monocomponent or mixtures of typically 2 or 3 different organic acids.Examples of organic acids are propionic acid, formic acid, citric acid,lactic acid, sorbic acid, malic acid, acetic acid, fumaric acid, benzoicacid, butyric acid and tartaric acid or their salt (typically sodium orpotassium salt such as potassium diformate or sodium butyrate).

In one embodiment, the amount of organic acid is 0.001% to 10% by weightof the composition. Examples of commercial products are VevoVitall® (DSMNutritional Products), Amasil®, Luprisil®, Lupro-Grain®, Lupro-Cid®,Lupro-Mix® (BASF), n-Butyric Acid AF (OXEA) and Adimix Precision(Nutriad).

Premix

The incorporation of the composition of feed additives as exemplifiedherein above to animal feeds, for example poultry feeds, is in practicecarried out using a concentrate or a premix. A premix designates apreferably uniform mixture of one or more microingredients with diluentand/or carrier. Premixes are used to facilitate uniform dispersion ofmicro-ingredients in a larger mix. A premix according to the inventioncan be added to feed ingredients or to the drinking water as solids (forexample as water soluble powder) or liquids.

Amino Acids

The composition of the invention may further comprise one or more aminoacids. Examples of amino acids which are used in animal feed are lysine,alanine, beta-alanine, threonine, methionine and tryptophan. In oneembodiment, the amount of amino acid is 0.001% to 10% by weight of thecomposition.

Vitamins and Minerals

In another embodiment, the animal feed may include one or more vitamins,such as one or more fat-soluble vitamins and/or one or morewater-soluble vitamins. In another embodiment, the animal feed mayoptionally include one or more minerals, such as one or more traceminerals and/or one or more macro minerals.

Usually fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed.

Non-limiting examples of fat-soluble vitamins include vitamin A, vitaminD3, vitamin E, and vitamin K, e.g., vitamin K3.

Non-limiting examples of water-soluble vitamins include vitamin C,vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6,niacin, folic acid and panthothenate, e.g., Ca-D-panthothenate.

Non-limiting examples of trace minerals include boron, cobalt, chloride,chromium, copper, fluoride, iodine, iron, manganese, molybdenum, iodine,selenium and zinc.

Non-limiting examples of macro minerals include calcium, magnesium,phosphorus, potassium and sodium.

In one embodiment, the amount of vitamins is 0.001% to 10% by weight ofthe composition. In one embodiment, the amount of minerals is 0.001% to10% by weight of the composition.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

In a still further embodiment, the animal feed additive of the inventioncomprises at least one of the below vitamins, preferably to provide anin-feed-concentration within the ranges specified in the below Table 1(for piglet diets, and broiler diets, respectively).

TABLE 1 Typical vitamin recommendations Vitamin Piglet diet Broiler dietVitamin A 10,000-15,000 IU/kg feed 8-12,500 IU/kg feed Vitamin D31800-2000 IU/kg feed 3000-5000 IU/kg feed Vitamin E 60-100 mg/kg feed150-240 mg/kg feed Vitamin K3 2-4 mg/kg feed 2-4 mg/kg feed Vitamin B12-4 mg/kg feed 2-3 mg/kg feed Vitamin B2 6-10 mg/kg feed 7-9 mg/kg feedVitamin B6 4-8 mg/kg feed 3-6 mg/kg feed Vitamin B12 0.03-0.05 mg/kgfeed 0.015-0.04 mg/kg feed Niacin (Vitamin B3) 30-50 mg/kg feed 50-80mg/kg feed Pantothenic acid 20-40 mg/kg feed 10-18 mg/kg feed Folic acid1-2 mg/kg feed 1-2 mg/kg feed Biotin 0.15-0.4 mg/kg feed 0.15-0.3 mg/kgfeed Choline chloride 200-400 mg/kg feed 300-600 mg/kg feed

Other Feed Ingredients

The composition of the invention may further comprise colouring agents,stabilisers, growth improving additives and aroma compounds/flavourings,polyunsaturated fatty acids (PUFAs); reactive oxygen generating species,antioxidants, anti-microbial peptides, anti-fungal polypeptides andmycotoxin management compounds.

Examples of colouring agents are carotenoids such as beta-carotene,astaxanthin, and lutein.

Examples of aroma compounds/flavourings are creosol, anethol, deca-,undeca- and/or dodeca-lactones, ionones, irone, gingerol, piperidine,propylidene phatalide, butylidene phatalide, capsaicin and tannin.

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in WO 03/044049 and WO 03/048148, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and enzymes such as an oxidase,an oxygenase or a syntethase.

Antioxidants can be used to limit the number of reactive oxygen specieswhich can be generated such that the level of reactive oxygen species isin balance with antioxidants.

Mycotoxins, such as deoxynivalenol, aflatoxin, zearalenone and fumonisincan be found in animal feed and can result in negative animalperformance or illness. Compounds which can manage the levels ofmycotoxin, such as via deactivation of the mycotoxin or via binding ofthe mycotoxin, can be added to the feed to ameliorate these negativeeffects. Examples of mycotoxin management compounds are Vitafix®,Vitafix Ultra (Nuscience), Mycofix®, Mycofix® Secure, FUMzyme®, Biomin®BBSH, Biomin® MTV (Biomin), Mold-Nil®, Toxy-Nil® and Unike® Plus(Nutriad).

Uses Use in Animal Feed

A LYS polypeptide of the invention may also be used in animal feed,wherein the term “animal” refers to all animals except humans. Examplesof animals are mono-gastric animals, e.g. pigs or swine (including, butnot limited to, piglets, growing pigs, and sows); poultry (including butnot limited to poultry, turkey, duck, quail, guinea fowl, goose, pigeon,squab, chicken, broiler, layer, pullet and chick); fish (including butnot limited to amberjack, arapaima, barb, bass, bluefish, bocachico,bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid,cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami,grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish,mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike,pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner,sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish,sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleyeand whitefish); and crustaceans (including but not limited to shrimpsand prawns).

In the use according to the invention the LYS polypeptide can be fed tothe animal before, after, or simultaneously with the diet. The latter ispreferred.

In a particular embodiment, the LYS polypeptide, in the form in which itis added to the feed, or when being included in a feed additive, iswell-defined. Well-defined means that the LYS polypeptide preparation isat least 50% pure as determined by Size-exclusion chromatography (seeExample 12 of WO 01/58275). In other particular embodiments the LYSpolypeptide preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, orat least 95% pure as determined by this method.

A well-defined LYS polypeptide preparation is advantageous. Forinstance, it is much easier to dose correctly to the feed a LYSpolypeptide that is essentially free from interfering or contaminatingother lysozymes. The term dose correctly refers in particular to theobjective of obtaining consistent and constant results, and thecapability of optimizing dosage based upon the desired effect.

For the use in animal feed, however, the LYS polypeptide need not bepure; it may e.g. include other enzymes, in which case it could betermed a LYS polypeptide preparation.

The LYS polypeptide preparation can be (a) added directly to the feed,or (b) it can be used in the production of one or more intermediatecompositions such as feed additives or premixes that is subsequentlyadded to the feed (or used in a treatment process). The degree of puritydescribed above refers to the purity of the original LYS polypeptidepreparation, whether used according to (a) or (b) above.

Methods of Improving Animal Performance

In an embodiment, the present invention also relates to a method ofimproving the performance of an animal comprising administering to theanimal the animal feed or the animal feed additive of the invention.

In a preferred embodiment, the method of improving the performance of ananimal comprises administering to the animal the animal feed or theanimal feed additive comprising the LYS polypeptide of the invention. Inone embodiment, the LYS polypeptide is selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12,SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO:30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 and SEQID NO: 45.

In an embodiment, the present invention also relates to the use of theanimal feed or an animal feed additive of the invention for improvingthe performance of an animal. In another embodiment, the inventionrelates to the use of one or more lysozymes of the invention forimproving the performance of an animal.

In one embodiment, ‘improving the performance of an animal’ means thatthere is an increase in body weight gain. In another embodiment,‘improving the performance of an animal’ means that there is an improvedfeed conversion ratio. In a further embodiment, ‘improving theperformance of an animal’ means that there is an increased feedefficiency. In a further embodiment, ‘improving the performance of ananimal’ means that there is an increase in body weight gain and/or animproved feed conversion ratio and/or an increased feed efficiency.

In an embodiment, the the animal feed comprises plant based materialselected from the group consisting of legumes, cereals, oats, rye,barley, wheat, maize, corn, sorghum, switchgrass, millet, pearl millet,foxtail millet, soybean, wild soybean, beans, lupin, tepary bean,scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean(fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed(oilseed rape), rice, beet, cabbage, sugar beet, spinach, quinoa, orpea, in a processed form thereof (such as soybean meal, rapeseed meal)or any combination thereof.

Methods of Preparing an Animal Feed

In an embodiment, the present invention provides a method for preparingan animal feed comprising adding one or more LYS polypeptide of theinvention to one or more animal feed ingredients. Animal feedingredients include, but are not limited to concentrates (as definedherein), forage (as defined herein), enzymes, probiotic, vitamins,minerals and amino acids.

In a preferred embodiment, the method of preparing an animal feedcomprises mixing plant based material with the LYS polypeptide of theinvention. In one embodiment, the LYS polypeptide is selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO:12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 27, SEQ IDNO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 andSEQ ID NO: 45.

In an embodiment, the plant based material is selected from the groupconsisting of legumes, cereals, oats, rye, barley, wheat, maize, corn,sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean,wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjimbean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil,peanut, Spanish peanut, canola, rapeseed (oilseed rape), rice, beet,cabbage, sugar beet, spinach, quinoa, or pea, in a processed formthereof (such as soybean meal, rapeseed meal) or any combinationthereof.

Preferred Embodiments

Herein follows a list of preferred embodiments of the invention.

-   1. A composition comprising at least 0.01 mg of LYS polypeptide per    kilogram of composition, wherein the polypeptide (a) has lysozyme    activity and (b) comprises one or more LAD catalytic domains;    wherein the LAD catalytic domain gives a domT score of at least 180    when queried using a Profile Hidden Markov Model (HMM) prepared    using SEQ ID NOs: 46 to 187 and hmmbuild software program, suitably    wherein the query is carried out using hmmscan software program by    the Method of Determining the LAD Catalytic Domain by HMM.-   2. The composition of item 1, wherein the polypeptide further    comprises one or more lysozyme enhancing domains, wherein the    lysozyme enhancing domain gives a domT score of at least 100 when    queried using a Profile Hidden Markov Model prepared using SEQ ID    NOs: 188 to 316 and hmmbuild software program, and wherein the query    is carried out using the hmmscan software program by the Method of    Determining the Lysozyme Enhancing Domain.-   3. The composition of any of items 1 to 2, wherein    -   (a) the LAD catalytic domain comprises one or more motif I:        AG[I/L]AT[A/G][I/L][T/V]ES (SEQ ID NO: 317) and/or one or more        motif II V[G/A]XLCQXVQXSAYP (SEQ ID NO: 318); and/or    -   (b) the lysozyme enhancing domain comprises one or more motif        III: [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN] (SEQ ID NO:        319).-   4. A composition comprising one or more LYS polypeptides having    lysozyme activity, wherein the polypeptide is dosed at least 0.01 mg    of polypeptide per kilogram of composition and is selected from the    group consisting of:    -   (a) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 3;    -   (b) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 6;    -   (c) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 9;    -   (d) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 12;    -   (e) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 15;    -   (f) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 18;    -   (g) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 21;    -   (h) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 24;    -   (i) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 27;    -   (j) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 33;    -   (l) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 36;    -   (m) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 39;    -   (n) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 42;    -   (o) a polypeptide having at least 80% sequence identity to the        polypeptide of SEQ ID NO: 45;    -   (p) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID        NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO:        24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36,        SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45        positions;    -   (q) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o)        or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;    -   (r) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o)        or (p) and a N-terminal and/or C-terminal extension of up to 10        amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;        and    -   (s) a fragment of the polypeptide of (a), (b), (c), (d), (e),        (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having        lysozyme activity and having at least 90% of the length of the        mature polypeptide.-   5. The composition of item 4, wherein the polypeptide is selected    from the group consisting of:    -   (a) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 3;    -   (b) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 6;    -   (c) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 9;    -   (d) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 12;    -   (e) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 15;    -   (f) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 18;    -   (g) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 21;    -   (h) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 24;    -   (i) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 27;    -   (j) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 30;    -   (k) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 33;    -   (l) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 36;    -   (m) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 39;    -   (n) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 42;    -   (o) a polypeptide having at least 85% sequence identity to the        polypeptide of SEQ ID NO: 45; and    -   (p) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID        NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO:        24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36,        SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32 or 33 positions.-   6. The composition of item 4, wherein the polypeptide is selected    from the group consisting of:    -   (a) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 3;    -   (b) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 6;    -   (c) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 9;    -   (d) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 12;    -   (e) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 15;    -   (f) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 18;    -   (g) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 21;    -   (h) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 24;    -   (i) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 27;    -   (j) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 30;    -   (k) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 33;    -   (l) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 36;    -   (m) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 39;    -   (n) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 42;    -   (o) a polypeptide having at least 90% sequence identity to the        polypeptide of SEQ ID NO: 45; and    -   (p) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID        NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO:        24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36,        SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21 or 22 positions.-   7. The composition of item 4, wherein the polypeptide is selected    from the group consisting of:    -   (a) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 3;    -   (b) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 6;    -   (c) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 9;    -   (d) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 12;    -   (e) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 15;    -   (f) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 18;    -   (g) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 21;    -   (h) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 24;    -   (i) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 27;    -   (j) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 30;    -   (k) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 33;    -   (l) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 36;    -   (m) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 39;    -   (n) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 42;    -   (o) a polypeptide having at least 95% sequence identity to the        polypeptide of SEQ ID NO: 45; and    -   (p) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID        NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO:        24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36,        SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 45, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 positions.-   8. The composition of any of items 4 to 7, wherein the LYS    polypeptide comprises one or more motifs selected from the group    consisting of

(a) motif I: (SEQ ID NO: 317) AG[I/L]AT[A/G][I/L][T/V]ES; (b) motif II(SEQ ID NO: 318) V[G/A]XLCQXVQXSAYP; and (c) motif III: (SEQ ID NO: 319)[CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN].

-   9. The composition of any of items 1 to 8, wherein the polypeptide    is of fungal origin.-   10. The composition of any of items 1 to 9, wherein the polypeptide    is obtained or obtainable from the taxonomic phylum Ascomycota,    preferably the taxonomic subphylum Pezizomycotina.-   11. The composition of any of items 1 to 10, wherein the polypeptide    comprises or consists of amino acids1 to 226 of SEQ ID NO: 2, amino    acids 1 to 226 of SEQ ID NO: 3, amino acids 1 to 226 of SEQ ID NO:    5, amino acids 1 to 226 of SEQ ID NO: 6, amino acids 1 to 223 of SEQ    ID NO: 8, amino acids 1 to 223 of SEQ ID NO: 9, amino acids 1 to 304    of SEQ ID NO: 11, amino acids 1 to 304 of SEQ ID NO: 12, amino acids    1 to 228 of SEQ ID NO: 14, amino acids 1 to 228 of SEQ ID NO: 15,    amino acids 1 to 230 of SEQ ID NO: 17, amino acids 1 to 230 of SEQ    ID NO: 18, amino acids 1 to 230 of SEQ ID NO: 20, amino acids 1 to    230 of SEQ ID NO: 21, amino acids 1 to 232 of SEQ ID NO: 23, amino    acids 1 to 232 of SEQ ID NO: 24, amino acids 1 to 228 of SEQ ID NO:    26, amino acids 1 to 228 of SEQ ID NO: 27, amino acids 1 to 228 of    SEQ ID NO: 29, amino acids 1 to 228 of SEQ ID NO: 30, amino acids 1    to 226 of SEQ ID NO: 32, amino acids 1 to 226 of SEQ ID NO: 33,    amino acids 1 to 225 of SEQ ID NO: 35, amino acids 1 to 225 of SEQ    ID NO: 36, amino acids 1 to 225 of SEQ ID NO: 38, amino acids 1 to    225 of SEQ ID NO: 39, amino acids 1 to 304 of SEQ ID NO: 41, amino    acids 1 to 304 of SEQ ID NO: 42, amino acids 1 to 227 of SEQ ID NO:    44, or amino acids 1 to 227 of SEQ ID NO: 45.-   12. The composition of any of items 1 to 11 further comprising one    or more formulating agents.-   13. The composition of item 12, wherein the one or more formulating    agent is selected from the group consisting of glycerol, ethylene    glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium    chloride, sodium benzoate, potassium sorbate, sodium sulfate,    potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium    carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol,    lactose, starch, kaolin, maltodextrin, cyclodextrin, wheat, PVA,    acetate, phosphate and cellulose or any combination thereof.-   14. The composition of any of items 1 to 13 further comprising one    or more additional enzymes.-   15. The composition of item 14 wherein the one or more additional    enzymes is selected from the group consisting of acetyl xylan    esterase, alpha-amylase, beta-amylase, arabinofuranosidase,    cellobiohydrolases, cellulase, feruloyl esterase, galactanase,    alpha-galactosidase, beta-galactosidase, beta-glucanase,    beta-glucosidase, lipase, lysophospholipase, lysozyme, mannanase,    alpha-mannosidase, beta-mannosidase, phytase, phospholipase A1,    phospholipase A2, phospholipase C, phospholipase D, protease,    pullulanase, pectinase, pectin lyase, xylanase, beta-xylosidase, or    any combination thereof.-   16. The composition of any of items 1 to 15 further comprising one    or more microbes.-   17. The composition of item 16, wherein the one or more microbes is    selected from the group consisting of Bacillus subtilis, Bacillus    licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus    pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans,    Bacillus circulans, Bifidobacterium bifidum, Bifidobacterium    animalis, Bifidobacterium sp., Carnobacterium sp., Clostridium    butyricum, Clostridium sp., Enterococcus faecium, Enterococcus sp.,    Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus    farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri,    Lactobacillus salivarius, Lactococcus Lactis, Lactococcus sp.,    Leuconostoc sp., Megasphaera elsdenii, Megasphaera sp., Pediococsus    acidilactici, Pediococcus sp., Propionibacterium thoenii,    Propionibacterium sp. and Streptococcus sp. or any combination    thereof.-   18. The composition of any of items 1 to 17 further comprising one    or more components selected from the list consisting of:    -   one or more vitamins;    -   one or more minerals;    -   one or more amino acids;    -   one or more phytogenics;    -   one or more prebiotics;    -   one or more organic acids; and    -   one or more other feed ingredients.-   19. A granule comprising the composition of any of items 1 to 18.-   20. The granule of item 19 wherein the granule is coated.-   21. The granule of item 20 wherein the coating comprises a salt    and/or wax and/or a flour.-   22. An animal feed additive comprising the composition of any of    items 1 to 18 or the granule of any of items 19 to 21.-   23. An animal feed comprising plant based material and the    composition of any of items 1 to 18, the granule of any of items 19    to 21 or the animal feed additive of item 22.-   24. The animal feed of item 23, wherein the plant based material is    selected from the group consisting of legumes, cereals, oats, rye,    barley, wheat, maize, corn, sorghum, switchgrass, millet, pearl    millet, foxtail millet, soybean, wild soybean, beans, lupin, tepary    bean, scarlet runner bean, slimjim bean, lima bean, French bean,    Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut,    canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar beet,    spinach, quinoa, or pea, in a processed form thereof (such as    soybean meal, rapeseed meal) or any combination thereof.-   25. A pelleted animal feed comprising plant based material and the    composition of any of items 1 to 18, the granule of any of items 19    to 21 or the animal feed additive of item 22.-   26. The pelleted animal feed of item 25, wherein the plant based    material is selected from the group consisting of legumes, cereals,    oats, rye, barley, wheat, maize, corn, sorghum, switchgrass, millet,    pearl millet, foxtail millet, soybean, wild soybean, beans, lupin,    tepary bean, scarlet runner bean, slimjim bean, lima bean, French    bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish    peanut, canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar    beet, spinach, quinoa, or pea, in a processed form thereof (such as    soybean meal, rapeseed meal) or any combination thereof.-   27. A liquid formulation comprising the composition of any of items    1 to 18.-   28. The liquid formulation of item 27, wherein the LYS polypeptide    is dosed between 0.01% to 25% w/w of liquid formulation, preferably    0.05% to 20% w/w LYS polypeptide, more preferably 0.2% to 15% w/w    LYS polypeptide, more preferably 0.5% to 15% w/w LYS polypeptide or    most preferably 1.0% to 10% w/w LYS polypeptide.-   29. The liquid formulation of any of items 27 to 28, wherein the    formulation further comprises 20% to 80% w/w of polyol.-   30. The liquid formulation of item 29, wherein the polyol is    selected from the group consisting of glycerol, sorbitol, propylene    glycol (MPG), ethylene glycol, diethylene glycol, triethylene    glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, dipropylene    glycol, polyethylene glycol (PEG) having an average molecular weight    below about 600 and polypropylene glycol (PPG) having an average    molecular weight below about 600 or any combination thereof.-   31. The liquid formulation of any of items 27 to 30, wherein the    formulation further comprises 0.01% to 2.0% w/w preservative.-   32. The liquid formulation of item 31, wherein the preservative is    selected from the group consisting of sodium sorbate, potassium    sorbate, sodium benzoate and potassion benzoate or any combination    thereof.-   33. The liquid formulation of any of items 27 to 32 further    comprising one or more components selected from the list consisting    of:    -   one or more enzymes;    -   one or more microbes;    -   one or more vitamins;    -   one or more minerals;    -   one or more amino acids;    -   one or more phytogenics;    -   one or more prebiotics;    -   one or more organic acids; and    -   one or more other feed ingredients.-   34. A method of preparing an animal feed comprising applying the    liquid formulation of any of items 27 to 33 onto plant based    material.-   35. The method of item 34, wherein the liquid formulation is applied    via a spray.-   36. The method of any of items 34 to 35, wherein the plant based    material is selected from the group consisting of legumes, cereals,    oats, rye, barley, wheat, maize, corn, sorghum, switchgrass, millet,    pearl millet, foxtail millet, soybean, wild soybean, beans, lupin,    tepary bean, scarlet runner bean, slimjim bean, lima bean, French    bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish    peanut, canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar    beet, spinach, quinoa, or pea, in a processed form thereof (such as    soybean meal, rapeseed meal) or any combination thereof.-   37. The method of any of items 34 to 36, wherein the plant based    material is in pelleted form.-   38. A pelleted animal feed prepared using the method of any of items    34 to 37.-   39. A method of improving one or more performance parameters of an    animal comprising administering to one or more animals the    composition of any of items 1 to 18, the granule of any of items 19    to 21, the animal feed additive of item 22, the animal feed of any    of items 23 to 24, the pelleted animal feed of any of items 25 to 26    or 38 or the liquid formulation of any of items 27 to 33.-   40. The method of item 39 wherein the performance parameter is    selected from the list consisting of body weight gain (BWG),    European Production Efficiency Factor (EPEF) and Feed Conversion    Ratio (FCR) or any combination thereof.-   41. A method of preparing an animal feed, comprising mixing the    composition of any of items 1 to 18, the granule of any of items 19    to 21, the animal feed additive of item 22, the animal feed of any    of items 23 to 24, the pelleted animal feed of any of items 25 to 26    or 38 or the liquid formulation of any of items 27 to 33 with plant    based material.-   42. The method of item 41, wherein the plant based material is    selected from the group consisting of legumes, cereals, oats, rye,    barley, wheat, maize, corn, sorghum, switchgrass, millet, pearl    millet, foxtail millet, soybean, wild soybean, beans, lupin, tepary    bean, scarlet runner bean, slimjim bean, lima bean, French bean,    Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut,    canola, rapeseed (oilseed rape), rice, beet, cabbage, sugar beet,    spinach, quinoa, or pea, in a processed form thereof (such as    soybean meal, rapeseed meal) or any combination thereof.-   43. Use of composition of any of items 1 to 18, the granule of any    of items 19 to 21, the animal feed additive of item 22, the animal    feed of any of items 23 to 24, the pelleted animal feed of any of    items 25 to 26 or 38 or the liquid formulation of any of items 27 to    33:    -   in animal feed;    -   in animal feed additives;    -   in the preparation of a composition for use in animal feed;    -   for improving the nutritional value of an animal feed;    -   for increasing digestibility of the animal feed; and/or    -   for improving one or more performance parameters in an animal.-   44. An isolated polypeptide having lysozyme activity, selected from    the group consisting of:    -   (a) a polypeptide having at least 95%, e.g., at least 96%, at        least 97%, at least 98%, at least 99%, or 100% sequence identity        to the polypeptide of SEQ ID NO: 3;    -   (b) a polypeptide having at least 94%, e.g., at least 95%, at        least 96%, at least 97%, at least 98%, at least 99%, or 100%        sequence identity to the polypeptide of SEQ ID NO: 6;    -   (C) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        9;    -   (d) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        12;    -   (e) a polypeptide having at least 87%, e.g., at least 88%, at        least 89%, at least 90%, at least 91%, at least 92%, at least        93%, at least 94%, at least 95%, at least 96%, at least 97%, at        least 98%, at least 99%, or 100% sequence identity to the        polypeptide of SEQ ID NO: 15;    -   (f) a polypeptide having at least 81%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        18;    -   (g) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        21;    -   (h) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        24;    -   (i) a polypeptide having at least 87%, e.g., at least 88%, at        least 89%, at least 90%, at least 91%, at least 92%, at least        93%, at least 94%, at least 95%, at least 96%, at least 97%, at        least 98%, at least 99%, or 100% sequence identity to the        polypeptide of SEQ ID NO: 27;    -   (j) a polypeptide having at least 96.2%, e.g., at least 97%, at        least 97.5%, at least 98%, at least 98.5%, at least 99%, or 100%        sequence identity to the polypeptide of SEQ ID NO: 30;    -   (k) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        33;    -   (l) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        36;    -   (m) a polypeptide having at least 81%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        39;    -   (n) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        42;    -   (o) a variant of the polypeptide of SEQ ID NO: 3, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 positions;    -   (p) a variant of the polypeptide of SEQ ID NO: 6, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13        positions;    -   (q) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 9 and SEQ ID NO: 36, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 positions;    -   (r) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 24, SEQ        ID NO: 33 and SEQ ID NO: 42, wherein the variant has lysozyme        activity and comprises one or more amino acid substitutions,        and/or one or more amino acid deletions, and/or one or more        amino acid insertions or any combination thereof in 1, 2, 3, 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,        38, 39, 40, 41, 42, 43, 44 or 45 positions;    -   (s) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 15 and SEQ ID NO: 27, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29        positions;    -   (t) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 18 and SEQ ID NO: 39, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 positions;    -   (u) a variant of the polypeptide of SEQ ID NO: 30, wherein the        variant has lysozyme activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7 or 8 positions;    -   (v) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p),        (q), (r), (s), (t) or (u) and a N-terminal and/or C-terminal        His-tag and/or HQ-tag;    -   (w) a polypeptide comprising the polypeptide of (a), (b), (c),        (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p),        (q), (r), (s), (t) or (u) and a N-terminal and/or C-terminal        extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8,        9 or 10 amino acids; and    -   (x) a fragment of the polypeptide of (a), (b), (c), (d), (e),        (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r),        (s), (t) or (u) having lysozyme activity and having at least 90%        of the length of the mature polypeptide.-   45. The polypeptide according to item 44, wherein the polypeptide    comprises or consists of amino acids 1 to 316 of SEQ ID NO: 2, amino    acids 1 to 316 of SEQ ID NO: 3, amino acids 1 to 322 of SEQ ID NO:    4, amino acids 1 to 318 of SEQ ID NO: 6, amino acids 1 to 318 of SEQ    ID NO: 7, amino acids 1 to 326 of SEQ ID NO: 8, amino acids 1 to 316    of SEQ ID NO: 10, amino acids 1 to 316 of SEQ ID NO: 11, amino acids    1 to 324 of SEQ ID NO: 12, amino acids 1 to 316 of SEQ ID NO: 14,    amino acids 1 to 316 of SEQ ID NO: 15, amino acids 1 to 324 of SEQ    ID NO: 16, amino acids 1 to 316 of SEQ ID NO: 18, amino acids 1 to    316 of SEQ ID NO: 19, amino acids 1 to 324 of SEQ ID NO: 20, amino    acids 1 to 316 of SEQ ID NO: 22, amino acids 1 to 316 of SEQ ID NO:    23, amino acids 1 to 324 of SEQ ID NO: 24, amino acids 1 to 516 of    SEQ ID NO: 26, amino acids 1 to 516 of SEQ ID NO: 27, amino acids 1    to 524 of SEQ ID NO: 28, amino acids 1 to 317 of SEQ ID NO: 30,    amino acids 1 to 317 of SEQ ID NO: 31, amino acids 1 to 325 of SEQ    ID NO: 32, amino acids 1 to 316 of SEQ ID NO: 34, amino acids 1 to    316 of SEQ ID NO: 35, amino acids 1 to 324 of SEQ ID NO: 36, amino    acids 1 to 316 of SEQ ID NO: 38, amino acids 1 to 316 of SEQ ID NO:    39 or amino acids 1 to 324 of SEQ ID NO: 40.-   46. A polynucleotide encoding the polypeptide of any of items 44 to    45.-   47. A nucleic acid construct or expression vector comprising the    polynucleotide of item 46 operably linked to one or more control    sequences that direct the production of the polypeptide in an    expression host.-   48. A recombinant host cell comprising the polynucleotide of item 46    operably linked to one or more control sequences that direct the    production of the polypeptide.-   49. The recombinant host cell of item 48, wherein the host is a    filamentous fungus, such as Aspergillus, Trichoderma or Fusarium, or    a yeast, such as Pichia or Saccharomyces.

50. The recombinant host cell of item 49, wherein the host is anAspergillus, such as Aspergillus awamori, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae.

-   51. The recombinant host cell of item 49, wherein the host is a    Trichoderma, such as Trichoderma harzianum, Trichoderma koningii,    Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma    viride.-   52. The recombinant host cell of item 48, wherein the host is a    Bacillus such as Bacillus alkalophilus, Bacillus amyloliquefaciens,    Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus    coagulans, Bacillus firmus, Geobacillus stearothermophilus, Bacillus    lautus, Bacillus lentus, Bacillus licheniformis, Bacillus    megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus    subtilis, or Bacillus thuringiensis.-   53. A method of producing the polypeptide of any of items 44 to 45,    comprising:    -   (a) cultivating a cell, which in its wild-type form produces the        polypeptide, under conditions conductive for production of the        polypeptide; and    -   (b) recovering the polypeptide.-   54. A method of producing the polypeptide of any of items 44 to 45,    comprising:    -   (a) cultivating the recombinant host cell of any of items 48 to        52 under conditions conducive for production of the polypeptide;        and    -   (b) recovering the polypeptide.-   55. A transgenic plant, plant part or plant cell transformed with a    polynucleotide encoding the polypeptide of any of items 44 to 45.-   56. A whole broth formulation or cell culture composition comprising    a polypeptide of any of items 44 to 45.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

Examples Strains

Escherichia coli Top-10 strain purchased from Invitrogen (LifeTechnologies, Carlsbad, Calif., USA) was used to propagate ourexpression vectors encoding for LYS polypeptides.

Aspergillus oryzae strain MT3568 was used for heterologous expression ofthe LYS polypeptide encoding sequences. A. oryzae MT3568 is an amdS(acetamidase) disrupted gene derivative of Aspergillus oryzae JaL355 (WO2002/40694) in which pyrG auxotrophy was restored by disrupting the A.oryzae acetamidase (amdS) gene with the pyrG gene.

The fungal strain NN044175 was isolated from soil samples collected fromChina, in 1998 by the dilution plate method with PDA medium, pH7, 25 C.It was then purified by transferring a single conidium onto a PDA agarplate. The strain NN044175 was identified as Penicillium simplicissimum,based on both morphological characteristics and ITS rDNA sequence.

The fungal strain NN053742 was isolated from a soil sample collectedfrom Hubei province, China, in 2011 by the dilution plate method withPDA medium, at pH3, 25 C. It was then purified by transferring a singleconidium onto a PDA agar plate. The strain NN053742 was identified asPenicillium vasconiae, based on both morphological characteristics andITS rDNA sequence.

The fungal strain NN058285 was isolated from soil samples collected fromGuizhou Province, China, in 2014 by the dilution plate method with PDAmedium pH3. It was then purified by transferring a single conidium ontoa PDA agar plate. The strain NN058285 was identified as Talaromycesproteolyticus, based on both morphological characteristics and ITS rDNAsequence.

The fungal strain NN053333 was isolated from soil samples collected fromHunan province, China, in 2010 by the dilution plate method with PDAmedium, pH7, 25 C. It was then purified by transferring a singleconidium onto a PDA agar plate. The strain NN053333 was identified asAspergillus sp. XZ2668, based on both morphological characteristics andITS rDNA sequence.

The fungal strain NN058605 was from CBS with access number as CBS100492.The strain NN058605 was identified as Penicillium antarcticum, based onboth morphological characteristics and ITS rDNA sequence.

The fungal strain NN047528 was isolated from soil samples collected fromChina, in 1998 by the dilution plate method with YG medium, pH7, 37C. Itwas then purified by transferring a single conidium onto a PDA agarplate. The strain NN047528 was identified as Ovatospora brasiliensis,based on both morphological characteristics and ITS rDNA sequence.

The fungal strain NN054749 was isolated from soil samples collected fromTibet, China, in 2012 by the dilution plate method with PDA medium, pH7,10C. It was then purified by transferring a single conidium onto a PDAagar plate. The strain NN054749 was identified as Penicilliumwellingtonense, based on both morphological characteristics and ITS rDNAsequence.

The fungal strain NN054129 was isolated from soil samples collected fromGotland, Sweden in 2011 by the dilution plate method with Water agar,24C. It was then purified by transferring a single conidium onto a PDAagar plate. The strain NN054129 was identified as Penicilliumroseopurpureum, based on both morphological characteristics and ITS rDNAsequence.

The fungal strain NN058650 was isolated from soil samples collected fromGuizhou Province, China, in 2014 by the dilution plate method with PDAmedium pH3. It was then purified by transferring a single conidium ontoa PDA agar plate. The strain NN058650 was identified as Penicilliumvirgatum, based on both morphological characteristics and ITS rDNAsequence.

The fungal strain NN046949 was isolated from soil samples collected fromChina, in 1998 by the dilution plate method with YG medium, pH7, 37C. Itwas then purified by transferring a single conidium onto a PDA agarplate. The strain NN046949 was identified as Aspergillus niveus, basedon both morphological characteristics and ITS rDNA sequence.

The fungal strain NN057921 was obtained through a collaboration withProfessor Cai Lei in Institude of Microbiology, CAS, in 2014. The strainwas collected from China. It was identified as Chaetomium sp. ZY369,based on both morphological characteristics and ITS rDNA sequence.

The fungal strain NN058427 was isolated from soil samples collected fromGuizhou Province, China, in 2014 by the dilution plate method with PDAmedium pH3, 25C. It was then purified by transferring a single conidiumonto a PDA agar plate. The strain N NN058427 was identified asTalaromyces atricola, based on both morphological characteristics andITS rDNA sequence.

The fungal strain NN053773 was obtained through a collaboration withInstitude of Microbiology, CAS, in 2011. The strain was collected fromChina and isolated by the dilution plate method with PDA medium pH7,10C. It was then purified by transferring a single conidium onto a PDAagar plate. The strain NN053773 was identified as Trichocladium asperum,based on both morphological characteristics and ITS rDNA sequence.

The fungal strain NN058086 was isolated from soil samples collected fromGuizhou Province, China, in 2014 by the dilution plate method with PDAmedium pH3, 25C. It was then purified by transferring a single conidiumonto a PDA agar plate. The strain NN058086 was identified as Metarhiziumcarneum, based on both morphological characteristics and ITS rDNAsequence.

Strain Thielavia terrestris strain NRRL 8126 was purchased ATCC, andinoculated onto a PDA plate and incubated for 7 days at 37° C. in thedarkness. Mycelia and spores from the plate were inoculated into 500 mlshake flasks containing 100 mls of YPG medium. The flasks were incubatedfor 6 days at 37° C. with shaking at 150 rpm.

Media and Solutions

DAP4C-1 medium was composed of 0.5 g yeast extract, 10 g maltose, 20 gdextrose, 11 g magnesium sulphate heptahydrate, 1 g dipotassiumphosphate, 2 g citric acid monohydrate, 5.2 g potassium phosphatetribasic monohydrate, 1 mL Dowfax 63N10 (antifoaming agent), 2.5 gcalcium carbonate, supplemented with 1 mL KU6 metal solution, anddeionised water to 1000 mL.

KU6 metal solution was composed of 6.8 g ZnCl₂, 2.5 g CuSO₄.5H₂O, 0.13 gNiCl₂, 13.9 g FeSO₄.7H₂O, 8.45 g MnSO₄.H₂O, 3 g C₆H₈O₇.H₂O, anddeionised water to 1000 mL.

YP 2% glucose medium was composed of 10 g yeast extract, 20 gBacto-peptone, 20 g glucose, and deionised water to 1000 mL.

LB plates were composed of 10 g of Bacto-tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionised water to 1000mL.

LB medium was composed of 10 g of Bacto-tryptone, 5 g of yeast extract,and 10 g of sodium chloride, and deionised water to 1000 mL.

COVE-Sucrose-T plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 mL of COVE salt solution, and deionised water to 1000 mL. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and 10 mM acetamide, Triton X-100 (50 μL/500mL) were added.

COVE-N-Agar tubes were composed of 218 g Sorbitol, 10 g Dextrose, 2.02 gKNO₃, 25 g agar, 50 mL Cove salt solution, and deionised water up to1000 mL.

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCL, 26 gof KH₂PO₄, 50 mL of COVE trace metal solution, and deionised water to1000 mL.

COVE trace metal solution was composed of 0.04 g of Na₂B₄O₇.10H₂O, 0.4 gof CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionised water to 1000 mL.YPMmedium contained 1% of Yeast extract, 2% of Peptone and 2% of Maltose.

Example 1: Determination of Lysozyme Activity Using Reducing Ends Assay

The LYS polypeptide was diluted in phosphate buffer (5 mM citrate, 5 mMK₂HPO₄, 0.01% TritonX-100, pH 5.0) to 50 μg/mL in polypropylene tubes.The diluted LYS polypeptide was further diluted in a 96-wellpolypropylene microtiter plate to a concentration of 5.0 or 0.7 μg/mL inphosphate buffer (5 mM citrate, 5 mM K₂HPO₄, 0.01% TritonX-100, pH 5.0).In a polypropylene deepwell plate 50 μL of the LYS polypeptide dilutionwas mixed with 450 μL 1% Micrococcus lysodeikticus solution (lyophilizedMicrococcus lysodeikticus ATCC No. 4698 (Sigma M3770) in milli-Q water)and incubated at 40° C. with shaking (500 rpm) for 45 min. Afterincubation the deepwell plate was centrifuged (4000 g, 5 min) to pelletinsoluble material and 100 μL of the supernatant was mixed with 50 μL3.2M HCl in a 96-well PCR plate and incubated at 95° C. for 80 min. 50μL of 3.5 M NaOH was added to each well of the PCR plate, and 150 μL ofeach sample was transferred to a new PCR plate containing 75 μL/well4-hydroxybenzhydrazide solution in K—Na tartrate/NaOH buffer (50 g/LK—Na tartrate+20 g/L NaOH). The plate was incubated at 95° C. for 10 minbefore 100 μL/sample was transferred to a clear flat-bottomed microtiterplate for optical density (OD) measurement at 405 nm. OD measurementswere performed on three times diluted samples (50 μL sample diluted in100 μL in Milli-Q water).

Example 2: Determination of Lysozyme Activity Using OD Drop Assay

Freeze-dried Micrococcus lysodeikticus ATCC No. 4698 (Sigma) was washedand suspended in 60 mM KH₂PO₄ buffer at pH6.0 with final concentrationof 1% (w/v) as substrate stock. The concentration of the strain wasadjusted by adding citric acid-Na₂HPO₄ buffer until OD450 reachapproximately 1.

Citric acid-Na₂HPO₄ pH4 buffer were prepared by adding 61.45 ml 0.1Mcitric acid and 38.55 ml 0.2M Na2HPO₄ for pH4. 20 μL enzyme at 50 μg/mLand 200 μL of diluted bacterial strain solution in citric acid-Na₂HPO₄buffer at pH4 were added to a 96 well plate, mixed and the OD450 wasread. Then the plate was incubated at 37° C., 300 rpm for 1 hour and theOD450 was read. The OD difference between the 1 hour time point to theinitial read showed the OD drop activity for the LYS polypeptide. Blankwas set by adding 20 ul MQ water or the corresponding buffer, and eachsample was measured in triplicate.

Example 3: Genomic DNA Extraction

Penicillium simplicissimum strain NN044175 was inoculated onto a PDAplate and incubated for 7 days at 25° C. in the darkness. Severalmycelia-PDA plugs were inoculated into 500 ml shake flasks containing100 ml of YPG medium. The flasks were incubated for 9 days at 25° C.with shaking at 160 rpm.

Penicillium vasconiae strain NN053742 was inoculated onto a PDA plateand incubated for 7 days at 25° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 3 days at 25° C. with shaking at160 rpm.

Talaromyces proteolyticus strain NN058285 was inoculated onto a PDAplate and incubated for 7 days at 25° C. in the darkness. Severalmycelia-PDA plugs were inoculated into 500 ml shake flasks containing100 ml of YPG medium. The flasks were incubated for 4 days at 25° C.with shaking at 160 rpm.

Aspergillus sp. strain NN053333 was inoculated onto a PDA plate andincubated for 7 days at 25° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 5 days at 25° C. with shaking at160 rpm.

Penicillium antarcticum strain NN058605 was inoculated onto a PDA plateand incubated for 7 days at 25° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 4 days at 25° C. with shaking at160 rpm.

Ovatospora brasiliensis strain NN047528 was inoculated onto a PDA plateand incubated for 7 days at 37° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 2 days at 37° C. with shaking at160 rpm.

Penicillium wellingtonense strain NN054749 was inoculated onto a PDAplate and incubated for 7 days at 25° C. in the darkness. Severalmycelia-PDA plugs were inoculated into 500 ml shake flasks containing100 ml of YPG medium. The flasks were incubated for 11 days at 25° C.with shaking at 160 rpm.

Penicillium roseopurpureum strain NN054129 was inoculated onto a PDAplate and incubated for 7 days at 25° C. in the darkness. Severalmycelia-PDA plugs were inoculated into 500 ml shake flasks containing100 ml of YPG medium. The flasks were incubated for 4 days at 25° C.with shaking at 160 rpm.

Penicillium virgatum strain NN058650 was inoculated onto a PDA plate andincubated for 7 days at 25° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 4 days at 25° C. with shaking at160 rpm.

Aspergillus niveus strain NN046949 was inoculated onto a PDA plate andincubated for 7 days at 25° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 3 days at 25° C. with shaking at160 rpm.

Chaetomium sp. strain NN057921 were inoculated onto a PDA plate andincubated for 7 days at 37° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 8 days at 37° C. with shaking at160 rpm.

The mycelia of Penicillium antarcticum strain NN058605 were collected byfiltration through MIRACLOTH® (Calbiochem, La Jolla, Calif., USA) andfrozen under liquid nitrogen. Frozen mycelia were ground, by a mortarand a pestle, to a fine powder, and genomic DNA was isolated using MPFast DNA spin kit for soil (MP Biomedicals, Santa Ana, Calif., USA)following the manufacturer's instruction.

Talaromyces atricola strain NN058427 was inoculated onto a PDA plate andincubated for 7 days at 25° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 3 days at 25° C. with shaking at160 rpm.

Trichocladium asperum strain NN053773 was inoculated onto a PDA plateand incubated for 7 days at 15° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 4 days at 15° C. with shaking at160 rpm.

Metarhizium carneum strain NN058086 was inoculated onto a PDA plate andincubated for 7 days at 25° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 4 days at 25° C. with shaking at160 rpm.

The mycelia of Thielavia terrestris were collected by filtration throughMIRACLOTH® (Calbiochem, La Jolla, Calif., USA) and frozen under liquidnitrogen. Frozen mycelia were ground, by a mortar and a pestle, to afine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit(24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer'sinstructions.

The mycelia of all the other strains were collected by filtrationthrough MIRACLOTH® and frozen under liquid nitrogen. Frozen mycelia wereground, by a mortar and a pestle, to a fine powder, and genomic DNA wasisolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden,Germany) following the manufacturer's instruction.

Example 4: Genome Sequencing, Assembly and Annotation

The extracted genomic DNA samples of Penicillium simplicissimum strainNN044175 were delivered to Exiqon A/S (Denmark) for genome sequencingusing an ILLUMINA® MiSeq System (Illumina, Inc., San Diego, Calif.,USA). The raw reads were assembled at Novozymes Denmark using programldba (Peng, Yu et al., 2010, Research in Computational MolecularBiology, 6044:426-440. Springer Berlin Heidelberg).

The extracted genomic DNA samples of Talaromyces proteolyticus strainNN058285, Penicillium antarcticum strain NN058605, Penicilliumroseopurpureum strain NN054129, Penicillium virgatum strain NN058650,Aspergillus niveus strain NN046949, Metarhizium carneum strain NN058086were delivered to Exiqon A/S for genome sequencing using an ILLUMINA®MiSeq System. The raw reads were assembled at Novozymes Denmark usingprogram Spades (Anton Bankevich et al., 2012, Journal of ComputationalBiology, 19(5): 455-477).

The extracted genomic DNA samples of Penicillium vasconiae strainNN053742, Ovatospora brasiliensis strain NN047528, Trichocladium asperumstrain NN053773 were delivered to Fasteris (Switzerland) for genomesequencing using an ILLUMINA® HiSeq 2000 System (Illumina, Inc., SanDiego, Calif., USA). The raw reads were assembled at Novozymes Denmarkusing program ldba.

The extracted genomic DNA samples of Aspergillus sp. strain NN053333,Chaetomium sp. strain NN057921 and Talaromyces atricola strain NN058427were delivered to Novozymes Davis (USA) for genome sequencing using anILLUMINA® MiSeq System. The raw reads were assembled at NovozymesDenmark using program Spades.

The extracted genomic DNA samples of Penicillium wellingtonense strainNN054749 were delivered to Novozymes Davis for genome sequencing usingan ILLUMINA® MiSeq System. The raw reads were assembled at NovozymesDenmark using program ldba.

The assembled sequences were analyzed using standard bioinformaticsmethods for gene identification and function prediction. GeneMark-ESfungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12):1979-1990) was used for gene prediction. Blastall version 2.2.10(Altschul et al., 1990, Journal of Molecular Biology. 215(3): 403-410,ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/2.2.10/) and HMMERversion 2.1.1 (National Center for Biotechnology Information (NCBI),Bethesda, Md., USA) were used to predict function based on structuralhomology. The NZ5 family was identified directly by analysis of theBlast results. The Agene program (Munch and Krogh, 2006, BMCBioinformatics 7: 263) and SignalP program (Nielsen et al., 1997,Protein Engineering 10: 1-6) were used to identify start codons. SignalPprogram was further used to predict signal peptides. Pepstats (Rice etal., 2000, Trends in Genetics. 16(6): 276-277) was used to predictisoelectric points and molecular weights.

Example 5: Cloning, Expression and Fermentation of Fungal NZ5 Genes (SEQID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37 and 40)

Fourteen fungal LYS wild type genes, LYS_Pesi (SEQ ID NO:1), LYS_Pv (SEQID NO:4), LYS_Tapr (SEQ ID NO:7), LYS_Asp2668 (SEQ ID NO:10), LYS_Pean(SEQ ID NO:13), LYS_chbr (SEQ ID NO:16), LYS_Pewe (SEQ ID NO:19), LYS_Pr(SEQ ID NO:22), LYS_Pevir (SEQ ID NO:25), LYS_asni (SEQ ID NO:28),LYS_ch369 (SEQ ID NO: 31), LYS_Taat (SEQ ID NO:34), LYS_Tras (SEQ ID NO:37), LYS_Meca2 (SEQ ID NO:40) were cloned from Penicilliumsimplicissimum strain NN044175, Penicillium vasconiae strain NN053742,Talaromyces proteolyticus strain NN058285, Aspergillus sp. strainNN053333, Penicillium antarcticum strain NN058605, Ovatosporabrasiliensis strain NN047528, Penicillium wellingtonense strainNN054749, Penicillium roseopurpureum strain NN054129, Penicilliumvirgatum strain NN058650, Aspergillus niveus strain NN046949, Chaetomiumsp. strain NN057921, Talaromyces atricola strain NN058427, Trichocladiumasperum strain NN053773, Metarhizium carneum strain NN058086respectively.

The fungal LYS genes were cloned into an Aspergillus oryzae expressionvector pCaHj505 as described in WO2013029496. The transcription of theLYS coding sequence with the native secretion signal was under thecontrol of an Aspergillus oryzae alpha-amylase gene promoter.

The final expression plasmids, p505-LYS_Pesi, p505-LYS_Pv,p505-LYS_Tapr, p505-LYS_Asp2668, p505-LYS_Pean, p505-LYS_chbr,p505-LYS_Pewe, p505-LYS_Pr, p505-LYS_Pevir, p505-LYS_asni,p505-LYS_ch369, p505-LYS_Taat, p505-LYS_Tras and p505-LYS_Meca2, wereindividually transformed into an Aspergillus oryzae expression host. TheLYS genes were integrated by homologous recombination into theAspergillus oryzae host genome upon transformation. Four transformantsof each transformation were selected from the selective media agar plateand inoculated to 3 ml of YPM or Dap4C medium in 24-well plate andincubated at 30° C., 150 rpm. After 3 days incubation, 20 μl ofsupernatant from each transformant were analyzed on NuPAGE Novex 4-12%Bis-Tris Gel w/MES according to the manufacturer's instructions. Theresulting gel was stained with Instant Blue. SDS-PAGE profiles of thecultures showed that all genes were expressed with 1 protein banddetected at approximately 28 kDa, 25 kDa, 25 kDa, 35 kDa, 25 kDa, 25kDa, 25 kDa, 25 kDa, 25 kDa, 25 kDa, 25 kDa, 25 kDa, 25 kDa, 30 kDa. Therecombinant Aspergillus oryzae strains with the strongest protein bandwere selected for shaking flask culturing. The recombinant strains wereinoculated on slant made of slant medium and incubated at 37C for 6-7days. When strains were well grown to fully sporulated, they wereinoculated to 2 L shaking flasks each containing 400 ml of YPM or DAP4C,5-6 flasks for each strain. Flasks were shaking at 80 rpm, 30C. Cultureswere harvested on day 3 or day 4 and filtered using a 0.22 μm DURAPOREMembrane and were purified as described in example 9.

Example 6: Construction of the Improved Split-Marker Aspergillus oryzaeHost

An improved Aspergillus oryzae host/vector system comparable to the onedescribed in example 5 disclosed in WO 2016026938A1 was constructed. Theimprovement was made to reduce the size of the transforming DNA bymoving the FLPase expression cassette located on PART-11 of the plasmidpDAu724 (see page 34 in WO2016/026938, FIG. 7 and SEQ ID NO:30) to theintegration locus amy2 in the genome of the host strain. The cloning ofthe FLPase expression cassette into pDAu703 (WO2016/026938 page 32 andFIG. 6 and SEQ ID:29) in done by amplification of the FLPase expressioncassette from pDAu724 and cloning in between FRT-F3 and the amdSselection marker of pDAu703 to give the plasmid pDAu770 (FIG. 1, SEQ IDNO: 320). The same protocol as described in WO2016/026938 page 33 wasused to transform the linearized plasmid pDAu770 into protoplasts of A.oryzae strain Ja11338 (disclosed in WO2012/160097). Transformants wereselected on AmdS selection plates to obtain strain DAu785. The resultingrecombinant host strain DAu785 has a modified amy2 locus comparable tothe one in DAU716 (WO2016/026938) with the addition of the FLPaseexpression cassette (FIG. 2, top panel). The host strain DAu785 is nowconstitutively expressing the FLPase site specific recombinase allowingthe integration at the FRT sites of the transforming DNA in this casethe PCR fragments obtained by Overlap Extension PCR reaction (FIG. 2,middle and bottom panels) and described in Example 7.

Example 7: Overlap Extension PCR Cloning (SEQ ID NO: 43)

A PCR amplification of SEQ ID NO: 43 encoding the LYS polypeptide wascarried out using Phusion High-Fidelity DNA polymerase (New EnglandBiolabs, BioNordika Denmark A/S, Herlev, Denmark) in a 50 μL volumereaction and the primers disclosed in table 2.

TABLE 2 PCR primers Primer SEQ Primer* ID NO: Sequence KKSC0972- 3215'-CTATATACACAACTGGGGATCCACC F ATGCAGCTCTCCCTCCTCGT KKSC0972- 3225'-TAGAGTCGACCCAGCCGCGCCGGCCA R TTACAACCCACCAGCCTGGC *-F-forward primer;-R-reverse primer; Bold letters represent coding sequence.

The PCR reaction mix consisted of 10 μL Phusion reaction buffer HF (5×);1 μL of PCR nucleotide Mix (10 mM); 2 μL forward cloning primers (2.5mM); 2 μL reverse cloning primers (2.5 mM); 1 μL Phusion High-FidelityDNA Polymerase # M0530L (2000U/mL); and PCR grade water up to 50 μL. PCRreaction was incubated on a thermocycler T100 (Biorad, Hercules, Calif.,USA) using the following program: initial denaturation of 2 min at 98°C. followed by 30 cycles of 10 sec at 98° C., 2 min at 72° C. and endingup by a final elongation of 10 min at 72° C. The PCR amplicon waspurified using AM Pure XP beads system kit (Agencourt, Beverly, Mass.,USA) adapted on a Biomek FXp Liquid handler (Beckman Coulter, Brea,Calif., USA).

pDAu724 plasmid was used as DNA template to amplify two PCR products (F1and F3) in reactions composed of 10 μL of KAPA polymerase buffer 5x, 1μL 10 mM KAPA PCR Nucleotide Mix, 1 μL of 10 μM of the appropriateforward primers (SEQ ID NO: 323 for F1 and SEQ ID NO: 325 for F3, table3), 1 μL of 10 μM of the appropriate reverse primers (SEQ ID NO: 324 forF1 and SEQ ID NO: 326 for F3, table 3), 1 to 10 ng of pDAu724 plasmid, 1μL of KAPA Biosystems polymerase KK2502 (1unit) and PCR-grade water upto 50 μL.

PCR amplification reactions were carried out on a DYAD® Dual-BlockThermal Cycler (MJ Research Inc., Waltham, Mass., USA) programmed for 2min. at 98° C. and followed by 35 cycles of 10 sec. at 98° C. and 2 min.at 72° C. and one final cycle of 10 min. at 72° C.

Five μl of the PCR reaction were analyzed by 1% agarose gelelectrophoresis using TAE buffer where DNA bands of the appropriate sizewere observed. The remaining PCR reactions were purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

TABLE 3 PCR primers Primer SEQ Primer ID NO: Sequence Forward 323GAATTCGAGCTCGGTACCTTGAAGTTC primer F1 Reverse 324GGTGGATCCCCAGTTGTGTATATAGAGGATT primer F1 Forward 325TGCGCGGCGCGGCTGGGTCGACTCTA primer F3 Reverse 326TTCACACAGGAAACAGCTATGACCATG primer F3

Overlap Extension PCR reaction for cloning the LYS polypeptide geneamplified from Thielavia terrestris gDNA was composed of 10 μL KAPApolymerase buffer (5X), 1 μL 10 mM KAPA PCR Nucleotide Mix, 50 ng of PCRfragment F1 and equimolar amounts of PCR fragment F3 and LYS polypeptidegene encoding for SEQ ID NO: 45, 1 μl KAPA Biosystems polymerase KK2502(1unit) and PCR-grade water up to 48 μL. Reaction was incubated on aDYAD® Dual-Block Thermal Cycler (MJ Research Inc., Waltham, Mass., USA)using a program composed of 2 min. at 98° C.; followed by 5 cycles eachcomposed of 10 sec. at 98° C., 30 sec. at 68° C., and 5 min. at 72° C.and completed by a final extension of 8 min. at 72° C.

During the OE PCR reaction, annealing between fragment F1 and the LYSpolypeptide gene encoding for SEQ ID NO: 45 was ensured by the overlapin SEQ ID NO: 327 included in the forward cloning primer (KKSC0972-F)and annealing between fragment F3 and the LYS polypeptide gene encodingfor SEQ ID NO: 45 was ensured by the overlapping SEQ ID NO: 328 includedin the reverse cloning primer (KKSC0972-R).

One μL of 10 mM primer SEQ1 and 1 μL of 10 mM primer SEQ4 were added tothe OE PCR reaction and the reaction was incubated a second time on aDYAD® Dual-Block Thermal Cycler (MJ Research Inc., Waltham, Mass., USA)using a program composed of 2 min at 98° C.; followed by 25 cycles eachcomposed of 10 sec. at 98° C., and 4 min. at 72° C. and completed by afinal extension of 10 min. at 72° C.

Five μl of the PCR reaction was analysed by 1% agarose gelelectrophoresis using TAE buffer where an DNA band of the appropriatesize was observed. The remaining PCR reaction was up-concentrated to 20μL by heating the tube at 60° C. 10 μL of this reaction was used forAspergillus oryzae DAu785 protoplasts transformation.

Primer bind forward SEQ ID NO: 327: CTATATACACAACTGGGGATCCACCPrimer bind reverse SEQ ID NO: 328: TAGAGTCGACCCAGCCGCGCCGGCCA

Example 8: Preparation of Aspergillus protoplasts

Protoplasts of Aspergillus oryzae MT3568 were prepared according to WO95/002043. One hundred μl of protoplasts were mixed with OE PCR fragmentKKSC0972 and 250 μL of 60% PEG 4000 (Applichem, Darmstadt, Germany)(polyethylene glycol, molecular weight 4,000), 10 mM CaCl₂, and 10 mMTris-HCl pH 7.5 and gently mixed. The mixtures were incubated at 37° C.for 30 minutes and the protoplasts were spread onto COVE plates forselection. After incubation for 4-7 days at 37° C. spores of fourtransformants were inoculated into 0.2 mL of YP+2% glucose or DAP4C-1medium in 96 well microtiter plates. After 4 days cultivation at 30° C.,the culture broths were analysed by SDS-PAGE to identify transformantsproducing the highest amounts of LYS polypeptide.

Spores of the best transformant from the transformation were spread ontoCOVE plates containing 0.01% TRITON® X-100 in order to isolate singlecolonies. The spreading was repeated twice in total on COVE platescontaining 10 mM sodium nitrate. Spores were then inoculated into 500 mLshake flasks containing 100 mL of YP+2% glucose and incubated for 4 daysat 30° C. with shaking at 100 rpm. Culture broths were harvested byfiltration using a 0.2 μm filter device and purified as described inExample 9.

Example 9: Purification of LYS Polypeptides Activity Detection forPurification Procedure

Freeze-dried bacterial strains Micrococcus lysodeikticus ATCC No. 4698(Sigma) and Exiguobacterium sp. (isolated from soil) were separatelywashed and suspended in 60 mM KH₂PO₄ buffer at pH6.0 with finalconcentration of 1% (w/v) as substrate stock. Before activity detection,the concentration of substrate was diluted into 0.035% which correlatesto OD450 about 0.7 by 60 mM KH2PO4 buffer at pH6.0 or pH 4.0. 10 ul ofpolypeptide sample (or 5 ul of sample with 5 ul of MQ water ifcontaining high concentration of salt) and 190 ul of 0.035% substratewere added into 96-well plate, and then read OD450. The plate wasincubated for 30 or 60 minutes, 300 rpm at room temperature or 37° C. inthe thermomixer. The plate was shaked 10 seconds and read OD450 again.The OD drop showed lysozyme activity. Blank is added 10 μl of 60 mMKH₂PO₄ at pH 6.0 or pH4.0 buffer, and each sample was measured induplicate if necessary.

Purification of SEQ ID NO: 3

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then dialyzed with 20 mM NaAc at pH4.5. The solutionwas filtered with 0.45 um filter and then loaded into SP Fast Flowcolumn (GE Healthcare) equilibrated with 20 mM NaAc at pH4.5. A gradientincrease of NaCl concentration was applied as elution buffer from zeroto 1M, and then the elution fractions and flow-through fraction werecollected to detect lysozyme activity. The fractions with lysozymeactivity were analyzed by SDS-PAGE, and then concentrated for furtherevaluation. The protein concentration was determined by Qubit® ProteinAssay Kit (Invitrogen, cat Q33212).

Purification of SEQ ID NO: 6

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then the precipitation was added water to adjustconductance to about 140 mS/cm. The solution was filtered with 0.45 umfilter and then loaded into HIC High Performance column (GE Healthcare)equilibrated with 20 mM PBS at pH8.0 with 1.2M (NH₄)₂SO₄ added. Agradient decrease of (NH₄)₂SO₄ concentration was applied as elutionbuffer from 1.2M to zero, and then elution fractions and flow-throughfraction were collected to detect lysozyme activity.

The flow-through and Fractions from 1 to 15 were collected andconductance was adjusted to 180 mS/cm, then reloaded into HIC columnequilibrated with 20 mM PBS at pH8.0 with 1.8M (NH₄)₂SO₄ added. Agradient decrease of (NH₄)₂SO₄ concentration was applied as elutionbuffer from 1.8M to zero, and then elution fractions and flow-throughfraction were collected to detect lysozyme activity. The fractions withlysozyme activity were analyzed by SDS-PAGE, pooled together, anddiafiltrated with 20 mM PBS at pH6.0. The protein concentration wasdetermined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).

Purification of SEQ ID NO: 9

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then the precipitation was added water to adjustconductance to about 170 mS/cm. The solution was filtered with 0.45 umfilter and then loaded into HIC High Performance column (GE Healthcare)equilibrated with 20 mM PBS at pH7.0 with 1.5M (NH₄)₂SO₄ added. Agradient decrease of (NH₄)₂SO₄ concentration was applied as elutionbuffer from 1.5M to zero, and then elution fractions and flow-throughfraction were collected to detect lysozyme activity. The fractions withlysozyme activity were analyzed by SDS-PAGE, pooled together, anddiafiltrated with 20 mM PBS at pH6.0. The protein concentration wasdetermined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).

Purification of SEQ ID NO: 12

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then the precipitation was added water to adjustconductance to about 185 mS/cm. The solution was filtered with 0.45 umfilter and then loaded into HIC High Performance column (GE Healthcare)equilibrated with 20 mM PBS at pH6.0 with 1.8M (NH₄)₂SO₄ added. Agradient decrease of (NH₄)₂SO₄ concentration was applied as elutionbuffer from 1.8M to zero, and then elution fractions and flow-throughfraction were collected to detect lysozyme activity. The fractions withlysozyme activity were analyzed by SDS-PAGE, pooled together, anddiafiltrated with 20 mM Bis-Tris at pH6.0.

The sample was loaded into a Mono Q column (GE Healthcare) equilibratedwith 20 mM Bis-Tris at pH6.0. A gradient increase of NaCl concentrationwas applied as elution buffer from zero to 1M, and then the elutionfractions and flow-through fraction were collected to detect lysozymeactivity. The fractions with lysozyme activity were analyzed bySDS-PAGE, and then concentrated for further evaluation. The proteinconcentration was determined by Qubit® Protein Assay Kit (Invitrogen,cat Q33212).

Purification of SEQ ID NO: 15

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then the precipitation was added water to adjustconductance to about 170 mS/cm. The solution was filtered with 0.45 umfilter and then loaded into Phenyl Fast Flow column (GE Healthcare)equilibrated with 20 mM PBS at pH6.0 with 1.5M (NH₄)₂SO₄ added. Agradient decrease of (NH₄)₂SO₄ concentration was applied as elutionbuffer from 1.5M to zero, and then elution fractions and flow-throughfraction were collected to detect lysozyme activity.

The flow-through and Fractions with lysozyme activity were collected andconductance was adjusted to 190 mS/cm, then reloaded into HIC columnequilibrated with 20 mM PBS at pH6.0 with 1.5M (NH₄)₂SO₄ added again. Agradient decrease of (NH₄)₂SO₄ concentration was applied as elutionbuffer from 1.5M to zero, and then elution fractions and flow-throughfraction were collected to detect lysozyme activity. The fractions withlysozyme activity were analyzed by SDS-PAGE, pooled together, anddiafiltrated with 20 mM PBS at pH6.0. The protein concentration wasdetermined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).

Purification of SEQ ID NO: 18

The culture supernatant from the expression of LYS_chbr (SEQ ID NO:16)was firstly precipitated with ammonium sulfate (80% saturation), thenthe precipitation was added water to adjust conductance to about 170mS/cm. The solution was filtered with 0.45 um filter and then loadedinto Phenyl Sepharose High Performance column (GE Healthcare)equilibrated with 20 mM PBS at pH6.0 with 1.8M (NH₄)₂SO₄ added. Agradient decrease of (NH₄)₂SO₄ concentration was applied as elutionbuffer from 1.8M to zero, and then elution fractions and flow-throughfraction were collected to detect lysozyme activity. The fractions withlysozyme activity were analyzed by SDS-PAGE, and fractions were pooledtogether, and diafiltrated with 20 mM PBS at pH6.0. The proteinconcentration was determined by Qubit® Protein Assay Kit (Invitrogen,cat Q33212).

Analysis by intact molecular weight (MAXIS II electrospray massspectrometer (Bruker Daltonik GmbH, Bremen, Del.)) showed that the majorproduct corresponded to amino acids 1 to 230 of SEQ ID NO: 18 (detectedmass 24128.35 Da, predicted mass 24128.21 Da) with a minor productcorresponded to amino acids 4 to 230 of SEQ ID NO: 18 (detected mass23768.79 Da, predicted mass 23768.16 Da).

Purification of SEQ ID NO: 329

The culture supernatant from the expression of LYS_chbr (SEQ ID NO:16)was firstly precipitated with ammonium sulfate (80% saturation), thendialyzed with 20 mM PBS at pH6.5. The solution was filtered with 0.45 umfilter and then loaded into Capto SP column (GE Healthcare) equilibratedwith 20 mM PBS at pH6.5. A gradient increase of NaCl concentration wasapplied as elution buffer from zero to 1M, and then the elutionfractions and flow-through fraction were collected to detect lysozymeactivity. The fractions with lysozyme activity were analyzed bySDS-PAGE, and then concentrated for further evaluation. The proteinconcentration was determined by Qubit® Protein Assay Kit (Invitrogen,cat Q33212).

Analysis by N-terminal sequencing (Applied Biosystems Precise Amino AcidSequencer Model 494) and intact molecular weight (MAXIS II electrospraymass spectrometer (Bruker Daltonik GmbH, Bremen, Del.)) showed that theN-terminal LED domain had been cleaved off leaving the LAD catalyticdomain and that the molecule had a heterogeneous N-terminal (see table4). The major product corresponded to residues 85-230 which is disclosedas SEQ ID NO: 329.

TABLE 4 N-terminal and intact molecular weigh determinationIntact Molecular Weight Applied Biosystems Residues ofN-terminal sequence SEQ ID NO: 18 M.Wt Calculated M.Wt. Observed IDGNLPGLN 88-230 15167.31 Da 15167.92 Da OK GKGNLPG 86-230 15352.54 Da15353.04 Da OK GGKGNLP 85-230 15409.59 Da 15410.06 Da OK

Purification of SEQ ID NO: 21

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then the precipitation was added water to adjustconductance to about 140 mS/cm. The solution was filtered with 0.45 umfilter and then loaded into Phenyl Fast Flow column (GE Healthcare)equilibrated with 20 mM NaAc at pH4.5 with 2M NaCl added. A gradientdecrease of NaCl concentration was applied as elution buffer from 2M tozero, and then elution fractions and flow-through fraction werecollected to detect lysozyme activity.

The flow-through and Fractions with lysozyme activity were collected andconductance was adjusted to 180 mS/cm, then reloaded into HIC columnequilibrated with 20 mM NaAc at pH4.5 with 4M NaCl added again. Agradient decrease of NaCl concentration was applied as elution bufferfrom 4M to zero, and then elution fractions and flow-through fractionwere collected to detect lysozyme activity. The fractions with lysozymeactivity and unbound sample were analyzed by SDS-PAGE, pooled together,and concentrated. The protein concentration was determined by Qubit®Protein Assay Kit (Invitrogen, cat Q33212).

Purification of SEQ ID NO: 24

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then the precipitation was added water to adjustconductance to about 170 mS/cm. The solution was filtered with 0.45 umfilter and then loaded into HIC High Performance column (GE Healthcare)equilibrated with 20 mM PBS at pH6.0 with 4M NaCl added. A gradientdecrease of NaCl concentration was applied as elution buffer from 4M tozero, and then elution fractions and flow-through fraction werecollected to detect lysozyme activity.

The flow-through and unbound sample with lysozyme activity werecollected and conductance was adjusted to 190 mS/cm, then reloaded intoHIC column equilibrated with 20 mM PBS at pH6.0 with 1.8M (NH₄)₂SO₄added again. A gradient decrease of (NH₄)₂SO₄ concentration was appliedas elution buffer from 1.8M to zero, and then elution fractions andflow-through fraction were collected to detect lysozyme activity. Thefractions with lysozyme activity were analyzed by SDS-PAGE, pooledtogether, and diafiltrated with 20 mM PBS at pH6.0. The proteinconcentration was determined by Qubit® Protein Assay Kit (Invitrogen,cat Q33212).

Purification of SEQ ID NO: 27

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then dialyzed with 20 mM NaAc at pH5.5. The solutionwas filtered with 0.45 um filter and then loaded into Capto SP column(GE Healthcare) equilibrated with 20 mM NaAc at pH5.5. A gradientincrease of NaCl concentration was applied as elution buffer from zeroto 1M, and then the elution fractions and flow-through fraction werecollected to detect lysozyme activity. The fractions with lysozymeactivity were analyzed by SDS-PAGE. The fractions with lysozyme activitywere pooled and concentrated, but degradation of sample was found.

The conductance of sample was adjusted to 200 mS/cm, then reloaded intoPhenyl High Performance column equilibrated with 20 mM PBS at pH6.0 with2.0M (NH₄)₂SO₄ added again. A gradient decrease of (NH₄)₂SO₄concentration was applied as elution buffer from 2.0M to zero, and thenelution fractions and flow-through fraction were collected to detectlysozyme activity. The fractions with lysozyme activity were analyzed bySDS-PAGE, pooled together, and diafiltrated with 20 mM PBS at pH6.0. Theprotein concentration was determined by Qubit® Protein Assay Kit(Invitrogen, cat Q33212).

Purification of SEQ ID NO: 30

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then dialyzed with 20 mM NaAc at pH4.5. The solutionwas filtered with 0.45 um filter and then loaded into Capto SP column(GE Healthcare) equilibrated with 20 mM NaAc at pH4.5. A gradientincrease of NaCl concentration was applied as elution buffer from zeroto 1M, and then the elution fractions and flow-through fraction werecollected to detect lysozyme activity. The fractions with lysozymeactivity were analyzed by SDS-PAGE, and then concentrated for furtherevaluation. The protein concentration was determined by Qubit® ProteinAssay Kit (Invitrogen, cat Q33212).

Purification of SEQ ID NO: 33

The culture supernatant of 033X73 was firstly precipitated with ammoniumsulfate (80% saturation), then dialyzed with 20 mM NaAc at pH4.5. Thesolution was filtered with 0.45 um filter and then loaded into Capto SPcolumn (GE Healthcare) equilibrated with 20 mM NaAc at pH4.5. A gradientincrease of NaCl concentration was applied as elution buffer from zeroto 1M, and then the elution fractions and flow-through fraction werecollected to detect lysozyme activity. The fractions with lysozymeactivity were analyzed by SDS-PAGE, and then concentrated for furtherevaluation. The protein concentration was determined by Qubit® ProteinAssay Kit (Invitrogen, cat Q33212).

Purification of SEQ ID NO: 36

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then dialyzed with 20 mM PBS at pH7.0. The solutionwas filtered with 0.45 um filter and then loaded into Capto Q column (GEHealthcare) equilibrated with 20 mM PBS at pH7.0. A gradient increase ofNaCl concentration was applied as elution buffer from zero to 1M, andthen the elution fractions and flow-through fraction were collected todetect lysozyme activity. The fractions with lysozyme activity wereanalyzed by SDS-PAGE. The flow-through fraction with lysozyme activitywas picked up for further purification.

The pH of flow-through fraction was adjusted to pH4.5, then reloadedinto Capto SP column equilibrated with 20 mM NaAC at pH4.5. A gradientincrease of NaCl concentration was applied as elution buffer from zeroto 1M, and then the elution fractions and flow-through fraction werecollected to detect lysozyme activity. The fractions with lysozymeactivity were analyzed by SDS-PAGE, and pooled together. The proteinconcentration was determined by Qubit® Protein Assay Kit (Invitrogen,cat Q33212).

Purification of SEQ ID NO: 39

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then the precipitation was added water to adjustconductance to about 170 mS/cm. The solution was filtered with 0.45 umfilter and then loaded into Phenyl Sepharose 6 Fast Flow column (GEHealthcare) equilibrated with 20 mM PBS at pH6.0 with 1.5M (NH₄)₂SO₄added. A gradient decrease of (NH₄)₂SO₄ concentration was applied aselution buffer from 1.5M to zero, and then elution fractions andflow-through fraction were collected to detect lysozyme activity. Thelysozyme activity still was found in flow-through fraction and fractions1 to 12, and they were pooled together for further purification.

The conductance of samples with lysozyme activity was adjusted to 190mS/cm, then reloaded into Phenyl Sepharose High Performance columnequilibrated with 20 mM PBS at pH6.0 with 1.8M (NH₄)₂SO₄ added again. Agradient decrease of (NH₄)₂SO₄ concentration was applied as elutionbuffer from 1.8M to zero, and then elution fractions and flow-throughfraction were collected to detect lysozyme activity. The fractions withlysozyme activity were analyzed by SDS-PAGE, pooled together, anddiafiltrated with 20 mM PBS at pH6.0. The protein concentration wasdetermined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).

Purification of SEQ ID NO: 42

The culture supernatant was firstly precipitated with ammonium sulfate(80% saturation), then the precipitation was added water to adjustconductance to about 170 mS/cm. The solution was filtered with 0.45 umfilter and then loaded into Phenyl Sepharose High Performance column (GEHealthcare) equilibrated with 20 mM PBS at pH6.0 with 1.5M (NH₄)₂SO₄added. A gradient decrease of (NH₄)₂SO₄ concentration was applied aselution buffer from 1.5M to zero, and then elution fractions andflow-through fraction were collected to detect lysozyme activity. Thefractions with lysozyme activity were analyzed by SDS-PAGE, but with twobands found. The fractions with lysozyme activity were pooled togetherfor further purification.

The conductance of the fractions was adjusted to 140 mS/cm, thenreloaded into Phenyl Sepharose High Performance column equilibrated with20 mM PBS at pH6.0 with 1.2M (NH₄)₂SO₄ added again. A gradient decreaseof (NH₄)₂SO₄ concentration was applied as elution buffer from 1.2M tozero, and then elution fractions and flow-through fraction werecollected to detect lysozyme activity. The fractions with lysozymeactivity were analyzed by SDS-PAGE. Fractions 29 to 37 have lowermolecular weight, were pooled together, and diafiltrated with 20 mM PBSat pH6.0. Fraction 43 to 45 have higher molecular weight, were pooledtogether, and diafiltrated with 20 mM PBS at pH6.0. The proteinconcentration was determined by Qubit® Protein Assay Kit (Invitrogen,cat Q33212).

Analysis by N-terminal sequencing (Applied Biosystems Precise Amino AcidSequencer Model 494) showed that the product began with the N-terminalsequence YPIKDNN, corresponding to amino acids 1 to 7 of SEQ ID NO: 42.

Analysis by intact molecular weight (MAXIS II electrospray massspectrometer (Bruker Daltonik GmbH, Bremen, Del.)) showed that the majorproduct corresponded to amino acids 1 to 304 of SEQ ID NO: 42 (detectedmass 31755.59 Da, predicted mass 31754.97 Da). There was also a smallamount of a secondary product corresponding to amino acids 76 to 304 ofSEQ ID NO: 42 (detected mass 23617.23 Da, predicted mass 23617.15 Da)due to the first LED domain being cleaved off the N-terminal.

Purification of SEQ ID NO: 45

The fermentation supernatant with the lysozyme was filtered through aFast PES Bottle top filter with a 0.22 μm cut-off. 250 ml filteredfermentation samples was diluted with 250 ml MilliQ water and pH wasadjusted to 4.5. The lysozyme containing solution was purified bychromatography on Capto S, approximately 30 ml in a XK16 column, usingas buffer A 50 mM Na-acetate pH 4.5, and as buffer B 50 mM Na-acetate+2M NaCl pH 4.5 using a 0-100% gradient over ca. 10CV. The fractions fromthe column were pooled based on the chromatogram (absorption at 280 and254 nm) and SDS-PAGE analysis.

The molecular weight was estimated to 25 kDa from SDS-PAGE and thepurity was >90%.

Example 10: Method of Determining the LAD Catalytic Domain by HMM

SEQ ID NOs: 46 to 187 were aligned using the software program MUSCLEv3.8.31 with the default settings. Using this alignment, the HMM wasconstructed using the software program ‘hmmbuild’ from the package HMMER3.0 (March 2010) (http://hmmer.org/) and the software was invoked usingthe default settings by the command: hmmscan3- -tblout output.datmodel.hmm sequences.fasta. The LAD catalytic domain HMM profile therebygenerated for subsequent loading into the software program ‘hmmscan’ isgiven below.

HMMER3/b [3.0 | March 2010] NAME LAD catalytic domain LENG 136 ALPHamino RF no CS no MAP yes DATE Fri Apr 21 12:03:08 2017 NSEQ 142 EFFN1.547058 CKSUM 201442427 STATS LOCAL MSV −10.1515 0.71110 STATS LOCALVITERBI −10.6276 0.71110 STATS LOCAL FORWARD −4.1803 0.71110 HMM A C D EF G H I K L M N P Q R S T V W Y m−>m m−>i m−>d i−>m i−>i d−>m d−>d COMPO2.28000 4.46955 2.96306 2.70047 3.44014 2.89264 3.73492 2.95902 2.728372.64684 3.53697 3.08243 3.38858 2.79348 2.98339 2.54635 2.85094 2.678604.50931 3.45344 2.68610 4.42256 2.77533 2.73152 3.46377 2.40496 3.725263.29372 2.67763 2.69331 4.24673 2.90332 2.73683 3.18173 2.89805 2.378752.77520 2.98532 4.58508 3.61512 0.86176 1.29948 1.18774 1.49367 0.254310.00000 * 1 2.70450 4.96091 2.44483 2.25748 4.38595 1.70411 3.767083.83155 2.65982 3.40989 4.23378 2.69810 3.83638 2.89968 3.15311 2.538222.77031 3.44613 5.62694 4.24971 17 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.02516 4.090934.81328 0.61958 0.77255 0.70021 0.68613 2 2.95798 4.37838 4.419793.85216 2.84679 4.06442 4.15724 2.49260 3.70306 1.06749 3.04032 4.021314.37743 3.87638 3.84752 3.36755 3.18313 2.46409 3.85059 2.85522 18 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.02269 4.19301 4.91535 0.61958 0.77255 0.78684 0.607493 2.64712 5.07695 2.31050 2.33245 4.40298 2.91636 3.64509 3.865962.37831 3.34917 4.16311 2.30868 3.81995 2.76486 2.86800 2.40011 2.437283.46575 5.56134 4.16658 19 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.02259 4.19722 4.919570.61958 0.77255 0.69335 0.69294 4 2.14335 5.06169 2.79203 2.152304.37029 3.13800 3.41689 3.83159 2.36004 3.35168 4.11530 2.86703 3.320332.51530 2.75969 2.34749 2.87496 3.40178 5.51777 4.12854 20 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.02150 4.24624 4.96858 0.61958 0.77255 0.57423 0.82813 52.32752 4.64440 3.12310 2.59357 3.83630 3.40168 3.74641 3.18648 2.507362.57706 3.73330 3.10307 3.85311 2.95065 2.73902 2.58368 2.21457 2.535795.18615 3.89627 21 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01979 4.32837 5.05072 0.619580.77255 0.64493 0.74380 6 2.80872 5.24372 2.92193 2.38964 4.591623.53977 3.72591 4.02013 2.14929 3.51532 4.32009 2.91909 3.77704 1.365712.64059 2.86001 3.11361 3.64412 5.64559 4.31503 22 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01973 4.33131 5.05366 0.61958 0.77255 0.64820 0.74021 7 2.625424.63457 2.55319 2.60028 3.82280 3.52902 3.75230 3.03815 2.62398 2.579752.90066 3.12772 3.91645 2.93691 3.05484 2.18187 2.58195 2.78875 5.178713.89049 23 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01973 4.33131 5.05366 0.61958 0.772550.62772 0.76315 8 2.35670 5.14104 2.59023 2.29666 4.47219 2.930103.62820 3.94710 2.04764 3.43912 4.18729 2.84545 3.83264 2.55019 2.412652.56554 2.74894 3.52457 5.58079 4.17986 24 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.019564.34008 5.06243 0.61958 0.77255 0.52419 0.89657 9 2.70329 4.599463.33128 2.77671 3.58063 3.60472 2.72765 2.94006 2.73677 2.77463 3.694582.14343 3.98982 3.07351 3.13881 2.83571 2.93476 2.52531 5.01301 2.5794425 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01861 4.38940 5.11175 0.61958 0.77255 0.571900.83115 10 0.58061 4.39851 3.89198 3.70803 4.66940 3.05741 4.710684.00280 3.76833 3.77981 4.64326 3.71756 3.68666 4.04155 4.00008 2.691793.01786 3.44176 6.02272 4.86740 26 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01861 4.389405.11175 0.61958 0.77255 0.57190 0.83115 11 2.28535 5.06565 2.884712.36748 4.19761 3.27471 3.62729 3.53322 2.10760 3.18026 4.11808 2.802473.71214 2.51474 2.31369 2.57926 2.86084 3.38794 5.52458 3.98224 27 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01861 4.38940 5.11175 0.61958 0.77255 0.55657 0.8513812 1.95678 4.83280 2.92821 2.43631 4.06399 2.95241 3.71153 3.305702.47325 3.04114 3.90855 3.04375 3.89352 2.78279 2.87396 2.61802 2.537412.71961 5.34398 4.01659 28 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01849 4.39559 5.117940.61958 0.77255 0.56314 0.84262 13 2.65884 4.25606 4.53713 3.959313.05007 4.05376 4.40064 1.21113 3.82028 2.25497 3.31657 4.10649 4.400204.01626 3.95164 3.36535 2.97633 1.88140 4.97279 3.28929 29 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01849 4.39559 5.11794 0.61958 0.77255 0.56314 0.84262 143.09624 4.49244 5.08148 4.54556 3.69891 4.61127 5.10139 1.09511 4.436872.08328 3.22577 4.70341 4.88972 4.64468 4.57704 3.97183 3.20262 1.335035.56774 4.38580 30 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01849 4.39559 5.11794 0.619580.77255 0.56314 0.84262 15 1.87078 5.06861 2.67006 2.34424 4.370453.16553 3.65393 3.82623 2.28226 3.35531 4.12231 2.87229 3.85367 2.703492.62439 2.49490 2.76661 3.35529 5.19663 4.14646 31 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01849 4.39559 5.11794 0.61958 0.77255 0.44025 1.03248 16 2.433664.90407 3.07169 1.93881 4.14929 3.51322 3.71660 3.38468 2.44374 3.142123.97548 3.04089 3.90532 2.38664 2.91529 2.71114 2.76736 2.33204 5.403524.06677 32 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01756 4.44649 5.16884 0.61958 0.772550.48576 0.95510 17 1.69111 4.40326 3.80425 3.41677 4.37951 1.198004.42086 3.72489 3.41793 3.46042 4.30219 3.40956 3.96367 3.70234 3.738592.67449 2.74965 2.61793 5.75300 4.54416 33 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.017564.44649 5.16884 0.61958 0.77255 0.48576 0.95510 18 2.67801 5.176033.05013 2.31240 4.51586 3.49631 3.67694 3.96645 1.74435 3.22387 4.226072.91297 3.91428 2.52932 2.01448 2.64975 2.92944 3.35475 5.59394 4.2325934 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01756 4.44649 5.16884 0.61958 0.77255 0.485760.95510 19 2.48895 5.18440 2.65300 2.21077 4.52329 3.29253 3.652714.00175 1.95386 3.48470 4.12914 2.89759 3.86079 2.50491 2.18622 2.570652.85720 3.57201 5.61837 4.21554 35 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01756 4.446495.16884 0.61958 0.77255 0.48576 0.95510 20 2.43351 4.88117 2.770742.22081 4.11935 3.46932 3.71513 3.38326 2.42344 2.62406 2.69401 3.042263.90131 2.82540 2.62123 2.56251 2.91163 3.14773 5.38359 3.90443 36 - -2.68618 4.42225 2.77517 2.73121 3.46348 2.40513 3.72495 3.29354 2.677412.69355 4.24690 2.90347 2.73740 3.18147 2.89801 2.37887 2.77520 2.985194.58477 3.61503 0.04246 3.32766 5.16884 0.48651 0.95390 0.48576 0.9551021 2.69712 5.18519 2.72756 2.39570 4.51082 1.98188 3.08098 3.976722.20957 3.47648 4.23700 2.82477 3.89080 2.76334 2.37850 2.67797 2.969223.56665 5.62338 4.23301 38 - - 2.68633 4.42243 2.77509 2.73132 3.463722.40492 3.72439 3.29372 2.67759 2.69347 4.24708 2.90365 2.73730 3.180932.89819 2.37883 2.77537 2.98501 4.58495 3.61521 0.10524 2.36234 5.168841.78389 0.18390 0.48576 0.95510 22 3.08450 4.42239 4.90061 4.320173.36955 4.33853 4.69926 1.58167 4.16773 1.61994 2.77727 4.43627 4.634354.05013 4.24780 3.66605 3.31710 1.41643 5.15040 3.90088 49 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01756 4.44649 5.16884 0.61958 0.77255 0.48576 0.95510 232.59988 4.75855 3.17066 2.72521 4.40766 2.14059 3.94805 3.84514 2.778023.44012 4.23907 3.03326 2.07393 2.98757 3.22451 2.01551 2.47915 3.415765.65635 4.33552 50 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01756 4.44649 5.16884 0.619580.77255 0.48576 0.95510 24 2.39242 5.12137 2.88795 2.13091 4.331913.46970 3.57957 3.90215 2.31739 3.35239 4.03851 2.90526 2.72477 2.723792.19066 2.50080 2.91724 3.45054 5.57111 4.18029 51 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01756 4.44649 5.16884 0.61958 0.77255 0.48576 0.95510 25 2.716745.18303 2.94981 2.39383 4.38247 3.46358 3.04209 3.88509 1.96661 3.381184.23031 2.98468 3.89750 2.14189 2.03986 2.68912 2.96595 3.57326 5.603564.22936 52 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01756 4.44649 5.16884 0.61958 0.772550.48576 0.95510 26 1.71921 4.45728 3.85035 3.71589 4.87129 0.764394.79418 4.28684 3.88233 4.01035 4.83765 3.73888 4.01975 4.11397 4.126962.72115 3.06866 3.62581 6.18704 5.04378 53 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.017564.44649 5.16884 0.61958 0.77255 0.48576 0.95510 27 2.59152 2.487814.45380 3.85081 3.15329 3.82710 4.15011 1.84112 3.67495 1.97419 3.000193.92506 4.19158 3.38567 3.69697 3.13011 2.91063 2.23021 3.68826 3.4412454 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01756 4.44649 5.16884 0.61958 0.77255 0.485760.95510 28 2.65209 3.96839 3.84226 3.11334 3.36636 3.72249 4.001572.32334 3.11605 2.17547 3.29883 3.58242 4.09639 2.77690 3.22956 2.960752.67088 1.76667 4.85107 3.53972 55 - - 2.68619 4.42226 2.77521 2.731243.46355 2.40514 3.72495 3.29355 2.67742 2.69356 4.24691 2.90341 2.737353.18147 2.89802 2.37888 2.77521 2.98519 4.58478 3.61490 0.03420 3.578045.16884 0.73477 0.65319 0.48576 0.95510 29 2.07224 4.45423 4.824174.28807 3.72902 4.39122 4.89895 1.15537 4.18214 2.34671 3.58878 4.469494.73704 4.42219 4.36253 3.74086 3.24252 1.52460 5.49002 4.28913 59 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01756 4.44649 5.16884 0.61958 0.77255 0.48576 0.9551030 1.15453 3.74267 3.77490 3.26721 3.89912 2.36201 4.18636 3.247273.21520 2.97973 3.85757 3.52789 3.99672 3.50932 3.54238 2.73423 2.923432.74165 5.32643 3.69760 60 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01756 4.44649 5.168840.61958 0.77255 0.48576 0.95510 31 2.84534 4.33315 4.79654 4.215173.00397 4.21925 4.58222 1.49254 4.06138 1.49784 3.23805 4.07937 4.540784.22140 4.14680 3.54186 3.21703 1.75514 5.07561 3.90804 61 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01756 4.44649 5.16884 0.61958 0.77255 0.48576 0.95510 321.19836 4.20261 3.80733 3.28440 3.79916 3.46684 4.16982 3.08236 3.218152.85747 2.79726 3.55364 4.02738 3.51320 3.53422 2.61082 2.45216 2.775055.24805 4.03169 62 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01756 4.44649 5.16884 0.619580.77255 0.48576 0.95510 33 2.67861 4.43286 3.96982 3.42894 3.657533.77789 4.27411 2.58096 3.33280 2.44008 3.15099 3.73934 4.23535 3.641183.63033 3.09698 1.23658 2.16899 5.22762 4.01619 63 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510 34 1.446204.53665 3.43775 2.81280 3.93249 2.34265 3.97126 3.24629 2.88857 2.990553.71572 3.31646 3.97140 3.21318 3.28442 2.63939 2.78238 2.66627 5.311394.05101 64 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01749 4.45077 5.17311 0.61958 0.772550.48576 0.95510 35 3.04606 4.42292 4.75886 4.16570 2.88260 4.220294.49891 2.34117 4.00836 1.03933 2.20271 4.29137 4.51739 4.12726 4.084993.42153 3.27084 2.49510 4.93841 3.26659 65 - 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- 2.68620 4.42244 2.775202.73124 3.46373 2.40506 3.72514 3.29342 2.67743 2.69374 4.24709 2.903232.73741 3.18123 2.89767 2.37906 2.77520 2.98503 4.58496 3.61522 0.095682.45835 5.17311 1.90622 0.16092 0.48576 0.95510 99 2.64610 4.192874.51782 3.92763 2.77711 3.97094 4.30822 1.78813 3.57005 2.08485 3.282444.04333 4.32451 3.95582 3.66707 3.27791 3.03016 1.43763 4.87494 3.69158175 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.02184 4.45077 4.61181 0.61958 0.77255 0.485760.95510 100 2.57644 5.19801 2.27538 2.38818 4.52814 3.35083 3.687194.00392 2.23129 3.50058 4.25504 2.58839 2.30709 2.79494 2.80539 2.415212.96353 3.58321 5.64771 4.24193 176 - - 2.68622 4.42229 2.77523 2.731273.46358 2.40516 3.72498 3.29358 2.67744 2.69359 4.24693 2.90350 2.737433.18150 2.89804 2.37890 2.77523 2.98522 4.58481 3.61363 0.19911 1.743845.16884 0.21958 1.62384 0.48146 0.96202 101 2.99688 5.49109 2.316932.31426 4.82026 1.50085 3.89510 4.31373 2.80362 3.81760 4.62442 1.943874.01089 2.75286 3.34734 2.82386 3.26700 3.89190 5.98283 4.52864 178 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510102 2.70284 4.52919 3.24032 2.96189 3.71775 3.56073 3.97139 3.179652.94172 2.87349 3.75897 3.21460 4.03711 3.27740 3.29933 2.53512 2.466222.91647 1.54333 3.85468 179 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.02978 4.45077 4.035810.61958 0.77255 0.48576 0.95510 103 2.61665 5.23073 2.48892 1.935644.56747 3.45738 3.61649 4.05178 2.24240 3.53134 4.27684 2.80050 3.640021.94619 2.84919 2.64239 2.89577 3.61752 5.66449 4.25113 180 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01770 4.43869 5.16104 0.61958 0.77255 0.50512 0.92491 1042.50715 5.12699 2.77810 2.33590 4.44582 3.30582 3.61595 3.91337 2.304263.35320 4.17496 2.61050 3.85772 2.65896 2.39675 2.29108 2.36063 3.503525.57542 3.76373 181 - - 2.68618 4.42225 2.77515 2.73124 3.46354 2.405133.72495 3.29354 2.67741 2.69355 4.24690 2.90347 2.73740 3.18147 2.898012.37884 2.77520 2.98519 4.58477 3.61503 0.03088 3.70193 5.16104 0.631980.75831 0.47385 0.97446 105 2.60454 4.24739 3.87526 3.29633 3.360553.57144 3.72308 2.65342 3.19345 2.14470 1.84807 3.60313 4.10632 3.358603.01033 3.00275 2.85663 2.18919 4.84814 3.64240 184 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510 106 2.391345.15060 2.35146 2.27357 4.47362 3.41528 3.68003 3.94208 2.43822 3.377424.20939 2.86965 2.78407 2.78921 2.79582 2.01297 2.86585 3.53191 5.608234.21105 185 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01749 4.45077 5.17311 0.61958 0.772550.48576 0.95510 107 2.62098 4.25013 3.87151 3.29482 3.36602 3.736203.93542 2.37557 2.91952 2.03620 3.23025 3.60470 2.54484 3.47456 3.426003.00516 2.82863 1.90641 4.85591 3.64926 186 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.017494.45077 5.17311 0.61958 0.77255 0.48576 0.95510 108 2.06052 4.406863.81765 3.48760 4.63190 1.17260 4.52999 4.05972 3.52970 3.73232 4.536593.59957 3.80588 3.79688 3.84625 2.40774 1.76640 3.46864 5.96429 4.76728187 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01749 4.45077 5.17311 0.61958 0.77255 0.485760.95510 109 2.30528 5.00504 2.79584 2.32132 4.28012 3.35093 3.684603.71938 2.30210 3.20302 4.06501 2.97029 3.87976 2.52168 2.83814 2.533152.26799 2.84934 4.56909 4.11805 188 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01749 4.450775.17311 0.61958 0.77255 0.48576 0.95510 110 1.74241 4.18228 4.150063.56681 3.06887 3.80169 3.83739 2.56132 3.44556 1.80992 3.28778 3.772344.16953 3.66514 3.62758 3.08801 2.93731 2.40159 3.19051 3.11867 189 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510111 1.15417 2.40052 3.43268 3.31342 3.94825 3.05617 4.23524 3.291343.27802 2.88728 3.90490 3.55144 3.99881 3.56368 3.59756 2.72050 2.931862.96489 5.37506 4.15892 190 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01749 4.45077 5.173110.61958 0.77255 0.48576 0.95510 112 3.26861 5.44238 2.97663 2.643984.75892 3.69012 4.06288 4.30148 2.66362 3.78982 4.71915 3.28643 4.239210.76296 2.96287 3.24618 3.53605 3.96409 5.86880 4.56259 191 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510 1131.91522 4.95047 2.97467 2.39757 4.20895 3.50202 3.70112 3.61309 2.148703.19155 3.18272 3.01748 3.89338 2.73359 2.84536 2.53242 2.55518 3.293915.43928 4.09037 192 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01749 4.45077 5.17311 0.619580.77255 0.48576 0.95510 114 3.29634 4.59962 5.14480 4.67781 3.938484.66858 5.40358 1.71382 4.56710 2.45181 3.72266 4.84032 5.02031 4.851914.75526 4.08284 3.33697 0.67655 5.91411 4.69288 193 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510 115 3.155675.44348 2.80271 2.49407 4.78415 3.60247 3.94943 4.28979 2.58206 3.788464.65575 2.96667 4.14360 0.91366 2.91447 3.11238 3.41462 3.91870 5.875744.52658 194 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01749 4.45077 5.17311 0.61958 0.772550.48576 0.95510 116 2.71563 4.90409 3.12545 2.55928 4.14921 2.852893.44680 3.05862 2.22913 3.15932 3.97583 3.07155 3.92680 2.53911 2.168702.75050 2.94327 2.44686 5.39558 4.07226 195 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.017494.45077 5.17311 0.61958 0.77255 0.48576 0.95510 117 2.19207 4.404843.86126 3.64147 4.76639 3.17370 4.68117 4.15367 3.70231 3.88127 4.701133.67352 3.97014 3.97418 3.96685 0.66193 2.77617 3.52234 6.10860 4.92572196 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01749 4.45077 5.17311 0.61958 0.77255 0.485760.95510 118 1.23716 4.99590 2.82777 2.30137 4.34451 3.20483 3.854833.76823 2.68928 3.36557 4.18982 3.06501 3.97138 2.91804 3.03321 2.759153.06515 3.41893 5.59981 4.25545 197 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01749 4.450775.17311 0.61958 0.77255 0.48576 0.95510 119 2.63152 4.27604 4.036483.47068 1.69703 3.81555 3.85315 2.68413 3.36748 2.44928 3.39118 3.318284.18646 3.53607 3.59165 3.09668 2.86423 2.46901 4.64999 2.19303 198 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510120 3.10050 4.93865 3.89438 3.80685 5.08138 2.93213 4.95457 4.685243.99850 4.30514 5.22080 4.02495 0.41952 4.31180 4.23729 3.27299 3.605474.11129 6.18106 5.18331 199 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01749 4.45077 5.173110.61958 0.77255 0.48576 0.95510 121 2.63821 5.16765 1.71131 2.061854.48005 3.20764 3.58490 3.94854 2.45253 3.45931 4.22038 2.80491 3.880692.79852 2.94579 2.60137 2.82264 3.54145 4.92801 3.93756 200 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510 1221.86376 4.90237 3.28241 2.68163 4.15144 3.62338 3.62925 3.54290 2.365762.72221 3.98901 3.18063 4.00712 2.90768 1.74335 2.86644 3.03069 3.245615.38278 4.04754 201 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01749 4.45077 5.17311 0.619580.77255 0.48576 0.95510 123 4.19177 5.36263 4.97401 4.78151 2.113944.76326 3.76878 3.89368 4.60093 3.15403 4.48829 4.44462 5.10497 4.552024.59461 4.21077 4.43309 3.83039 3.88657 0.47676 202 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01749 4.45077 5.17311 0.61958 0.77255 0.48576 0.95510 124 1.796355.14532 2.26839 2.39026 4.45744 3.24099 3.34639 3.84057 2.35322 3.436254.19753 2.81236 3.87618 2.60394 2.87095 2.67589 2.92156 3.51869 5.597114.13300 203 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.02128 4.45077 4.66879 0.61958 0.772550.48576 0.95510 125 2.49141 5.20122 2.74864 2.18402 4.54180 3.396803.60493 4.01898 1.78979 3.49991 4.24589 2.89607 3.84714 2.16259 2.636072.67773 2.87195 3.59004 5.63139 4.23074 204 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.023214.44704 4.48894 0.61958 0.77255 0.49180 0.94553 126 2.70259 4.782883.10316 2.62084 3.23014 3.55717 2.77689 3.39328 2.58605 3.02278 3.863713.04868 3.94378 2.53415 2.11061 2.76991 2.93283 3.10180 3.35180 3.35576205 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01765 4.44148 5.16383 0.61958 0.77255 0.500700.93168 127 2.64699 4.39217 3.56865 2.05763 3.29481 3.66295 3.917392.60538 2.95345 2.50897 2.59486 3.40402 4.04141 3.00636 3.30409 2.908672.78869 2.22561 4.97634 3.56924 206 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01765 4.441485.16383 0.61958 0.77255 0.50070 0.93168 128 2.15651 5.08647 2.662182.31635 4.38993 2.79836 3.66501 3.84743 2.38128 3.37411 4.13923 2.893712.92678 2.67029 2.83170 2.46722 2.70744 3.45474 4.50984 4.16121 207 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01765 4.44148 5.16383 0.61958 0.77255 0.50070 0.93168129 2.47139 5.13557 2.73837 1.94974 4.45770 3.46490 3.60456 3.856232.21863 2.88369 4.18268 2.94198 3.82238 2.40411 2.53877 2.63956 2.799593.31420 5.58153 4.18624 208 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01765 4.44148 5.163830.61958 0.77255 0.50070 0.93168 130 0.54170 4.41455 3.99487 3.837584.67181 3.21702 4.80217 3.94160 3.87765 3.78720 4.68767 3.78860 4.025744.15301 4.08310 2.61480 3.04914 3.40878 6.07441 4.88693 209 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.04858 4.44148 3.33421 0.61958 0.77255 0.50070 0.93168 1312.47060 5.10222 2.92302 2.23845 4.41518 3.31163 3.65256 3.83300 2.307643.39268 4.15289 2.86002 3.85601 2.39911 2.32759 2.51827 2.29611 3.305965.55412 4.16668 210 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.03356 4.41110 3.86983 0.619580.77255 0.54715 0.86416 132 2.26873 5.14785 2.83342 2.18702 4.478273.38243 3.63990 3.95273 2.07133 3.41537 4.19342 2.78217 3.84499 2.335112.64759 2.45499 2.67363 3.49865 5.58841 4.18815 211 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.03225 4.39603 3.94192 0.61958 0.77255 0.56894 0.83500 133 3.294754.59909 5.27958 4.75843 3.67853 4.82300 5.34776 1.05587 4.65842 1.736673.38036 4.93089 5.04836 4.83561 4.78636 4.20753 3.60004 1.39603 5.699754.55067 212 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.19685 4.38251 1.79460 0.61958 0.772550.58780 0.81091 134 2.59525 2.26255 4.26037 3.68225 3.24006 3.811484.16702 2.18927 3.54565 2.25647 3.24248 3.85052 4.18879 3.76123 3.702653.11792 2.92528 1.72461 4.80046 2.95881 213 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.042634.20800 3.61724 0.61958 0.77255 0.77092 0.62099 135 2.12293 5.067952.53477 2.27785 4.37789 3.30834 3.61326 3.84072 2.22403 3.35911 4.124212.69439 3.80736 2.57158 2.81142 2.37052 2.76704 3.41396 5.52424 4.13208214 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.05033 4.19589 3.38058 0.61958 0.77255 0.787250.60714 136 1.41298 4.54007 3.16784 2.67479 3.96764 3.28514 3.859793.32496 2.60972 2.92660 3.87024 3.16231 3.88002 3.04176 3.09126 2.660152.58267 3.00986 5.31055 4.04058 215 - - 2.68596 4.42247 2.77539 2.731433.46365 2.40403 3.72516 3.29358 2.67762 2.69335 4.24680 2.90368 2.737613.18168 2.89806 2.37907 2.77541 2.98531 4.58498 3.61491 0.511890.91469 * 1.07030 0.41993 0.00000 * //

Example 11: Method of Determining the Lysozyme Enhancing Domain by HMM

SEQ ID NOs: 188 to 316 were aligned using the software program MUSCLEv3.8.31 with the default settings. Using this alignment, the HMM wasconstructed using the software program ‘hmmbuild’ from the package HMMER3.0 (March 2010) (http://hmmer.org/) and the software was invoked usingthe default settings by the command: hmmscan3- -tblout output.datmodel.hmm sequences.fasta. The lysozyme enhancing domain HMM profilethereby generated for subsequent loading into the software program‘hmmscan’ is given below.

HMMER3/b [3.0 | March 2010] NAME lysozyme_enhancing_domain LENG 73 ALPHamino RF no CS no MAP yes DATE Tue Feb 3 15:29:15 2015 NSEQ 129 EFFN1.263702 CKSUM 3302514446 STATS LOCAL MSV −9.1036 0.71868 STATS LOCALVITERBI −9.7357 0.71868 STATS LOCAL FORWARD −3.7686 0.71868 HMM A C D EF G H I K L M N P Q R S T V W Y m−>m m−>i m−>d i−>m i−>i d−>m d−>d COMPO2.64236 3.16005 2.87141 2.79417 3.60706 2.63596 3.86157 2.94229 2.652792.95816 3.97690 3.11757 3.46392 3.12498 3.11011 2.56828 2.58627 2.580864.17029 3.04296 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.17958 4.32413 1.88959 0.61958 0.772550.00000 * 1 3.80107 5.04040 4.67499 4.39045 1.81828 4.48873 3.562853.50991 4.23379 2.82560 4.11380 4.16131 4.81340 4.22617 4.28030 3.879974.03091 3.43577 3.70270 0.73371 1 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.02327 4.167834.89017 0.61958 0.77255 0.67437 0.71228 2 2.33952 4.44593 3.238903.02979 4.35083 3.16321 4.20776 3.67603 3.02584 3.40031 4.31198 3.320211.10553 3.46227 3.39562 2.64660 2.86963 3.24503 5.70138 4.44714 2--2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.02327 4.16783 4.89017 0.61958 0.77255 0.58149 0.818863 2.99224 4.47028 5.00531 4.50059 3.72470 4.59877 5.18674 1.107014.40105 2.19903 3.51509 4.69439 4.89181 4.65354 4.58483 3.98375 3.447151.21838 5.67709 4.47722 3-- 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.02218 4.21548 4.937830.61958 0.77255 0.62351 0.76800 4 2.67119 4.76820 3.04982 2.541954.12069 3.43519 3.75916 3.51149 2.19874 3.13703 3.97428 2.96845 3.895582.93360 2.89915 2.57877 1.61288 3.10281 5.38435 4.08520 4-- 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.02491 4.21548 4.62159 0.61958 0.77255 0.52151 0.90048 52.37234 5.08464 2.49824 2.21575 4.41120 1.99198 3.58111 3.86677 2.528823.41126 4.20120 2.81325 3.86140 2.84200 3.02691 2.42476 2.83819 3.480015.60144 4.21122 5-- 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.02114 4.26301 4.98536 0.619580.77255 0.56183 0.84436 6 2.63301 5.17310 2.09015 2.17272 4.494633.27178 3.65545 3.97097 2.38584 3.47241 4.23433 2.52660 3.34330 2.768742.94084 2.41690 2.44683 3.55393 5.62559 4.21427 6-- 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.07099 4.26555 2.90975 0.61958 0.77255 0.56422 0.84119 7 2.611114.85146 2.70126 2.41033 4.02944 2.66646 3.65077 3.52514 2.43178 3.119343.92649 2.77281 3.83428 2.77300 2.91547 2.42718 2.41739 2.79685 5.354153.75427 7-- 2.68619 4.42226 2.77521 2.73124 3.46355 2.40511 3.724963.29355 2.67742 2.69356 4.24691 2.90348 2.73741 3.18147 2.89802 2.378882.77504 2.98519 4.58478 3.61504 0.09494 2.48394 4.93906 0.38374 1.143530.52245 0.89910 8 3.16563 4.52406 5.00410 4.47860 3.59477 4.613185.11093 1.75130 4.36399 1.66390 3.39589 4.68330 4.88103 4.58179 4.529243.98371 3.48258 0.98654 5.56065 4.39116 9-- 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.021084.26555 4.98790 0.61958 0.77255 0.56422 0.84119 9 2.74568 5.181032.62071 2.37499 4.50785 3.44148 3.43386 3.97009 2.14487 3.46738 4.240841.77122 3.86903 2.77625 2.44752 2.65464 2.97757 3.56615 5.60976 4.2264010 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.02108 4.26555 4.98790 0.61958 0.77255 0.439651.03356 10 3.21633 0.35479 4.79708 4.65548 4.57914 3.72194 5.207873.82938 4.49870 3.70850 4.85167 4.53077 4.45916 4.82584 4.53365 3.485563.72159 3.53505 5.86530 4.85631 11 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.324135.04648 0.61958 0.77255 0.48576 0.95510 11 3.30119 5.31389 3.736683.09114 4.51121 3.86969 3.13217 4.16018 2.10415 3.58751 4.49408 3.459924.26052 2.99924 0.83233 3.31264 3.47550 3.85705 5.50391 4.26079 12 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551012 2.24421 4.51545 3.30473 2.81428 4.39051 3.23670 4.14928 3.774913.04692 3.44962 4.27960 3.30747 3.89875 3.36729 3.42822 1.05895 2.514373.31859 5.70279 4.43964 13 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.046480.61958 0.77255 0.48576 0.95510 13 2.69468 4.75034 2.89429 2.563414.66767 0.94912 4.20148 4.12822 3.15097 3.75743 4.60332 3.20872 3.967573.42394 3.55601 2.47312 3.12631 3.62410 5.94365 4.63448 14 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 142.67424 4.69741 3.65691 3.53445 4.63811 3.40395 4.65858 4.09486 3.631503.77181 4.76683 3.75211 0.59051 3.99224 3.88152 2.99980 3.31792 3.644805.92609 4.77080 15 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.619580.77255 0.48576 0.95510 15 2.26323 4.51933 3.21961 2.94036 4.474111.38422 4.14822 3.91245 3.06085 3.54037 4.34588 2.97288 3.87499 3.352193.46854 1.96711 2.52323 3.40350 5.76384 4.49002 16 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 16 2.206684.36145 3.82003 3.56662 4.41076 3.20360 4.55272 3.51597 3.53768 3.413044.35967 3.64187 3.96341 3.86648 3.78744 2.68079 0.81042 3.10831 5.839314.64399 17 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.772550.48576 0.95510 17 2.61570 5.10849 2.62653 2.38205 4.43252 2.780783.60811 3.89018 2.47426 3.42628 4.20945 2.71231 3.87390 2.61734 2.999491.64993 2.94705 3.49936 5.60799 4.21799 18 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.019874.32413 5.04648 0.61958 0.77255 0.48576 0.95510 18 2.58292 4.586893.02231 2.68583 3.00245 3.32285 2.97170 3.14564 2.67302 2.81683 3.679392.99852 3.79428 3.00246 3.09283 2.65865 2.82740 2.88473 5.11794 2.2547819 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.485760.95510 19 2.02971 5.05961 2.68453 2.36019 4.37471 3.26819 3.654203.83090 2.10672 3.36092 4.13132 2.92980 3.45060 2.77060 2.82369 2.169052.88648 3.43886 5.53518 4.15201 20 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.324135.04648 0.61958 0.77255 0.48576 0.95510 20 3.16684 4.47509 4.981514.48359 3.78153 4.52597 5.13073 1.23357 4.37208 2.38350 3.60515 4.649474.86068 4.63385 4.54770 3.62858 3.43521 1.03896 5.65865 4.43929 21 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551021 2.67985 4.50015 3.40866 2.83947 3.66095 3.60615 3.61051 2.500382.12953 2.71143 3.59755 3.27678 3.98763 2.81347 3.06211 2.85030 2.836751.92294 5.06195 3.81377 22 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.046480.61958 0.77255 0.48576 0.95510 22 2.57540 5.16214 3.25351 2.633764.54584 3.62294 3.69980 3.93939 1.30635 3.43432 4.24813 3.13299 4.005442.78831 2.11440 2.89381 2.74985 3.58326 5.55169 4.27942 23 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 232.54455 4.73655 3.02733 2.59505 4.05985 3.44683 3.70095 3.46466 2.600563.09418 3.92399 3.08417 3.89235 2.67009 3.04116 2.11836 1.78482 2.959745.35882 4.04770 24 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.619580.77255 0.48576 0.95510 24 3.40982 4.75103 4.67192 4.22624 1.973084.35966 3.81417 3.04888 4.07862 2.38151 3.72579 4.16721 4.67838 4.147174.17038 3.68839 3.63576 2.47365 4.02768 0.99393 25 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 25 2.336075.05721 2.94517 2.24042 4.36369 3.44673 3.55543 3.81754 1.82729 3.319484.11325 2.80831 3.17764 2.75227 2.78214 2.58400 2.65726 3.32087 5.514794.13659 26 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.772550.48576 0.95510 26 2.36555 5.04925 3.04993 2.35049 4.35720 3.519893.68369 3.71551 1.45376 3.06828 4.12214 3.00561 3.91721 2.74243 2.724092.73518 2.95391 3.42066 5.49677 4.16815 27 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.019874.32413 5.04648 0.61958 0.77255 0.48576 0.95510 27 2.61202 4.800982.75023 2.62858 4.42906 1.32629 3.93435 3.86483 2.78346 3.46292 4.279883.09911 3.91159 3.10786 2.85440 2.43819 2.91018 3.44468 5.68001 4.3517828 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.485760.95510 28 2.39518 5.06692 2.42469 2.35324 4.15968 3.22653 3.048263.83188 2.39351 3.35617 4.12096 2.82671 3.83411 2.52507 2.87979 2.436222.35726 3.41027 5.52582 4.13893 29 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.324135.04648 0.61958 0.77255 0.48576 0.95510 29 2.56098 5.10975 1.847002.22610 4.41970 3.43432 3.64564 3.82090 2.31577 3.39892 4.16246 2.882693.84241 2.68525 2.69153 2.62766 2.81515 3.48193 5.56120 3.47482 30 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551030 2.71289 4.20867 4.05384 3.08944 3.32534 3.70711 4.11635 2.104603.37170 2.31376 3.31881 3.74286 4.18005 3.62760 3.59433 3.09939 2.947431.40821 4.85949 3.10435 31 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.046480.61958 0.77255 0.48576 0.95510 31 2.56066 3.98356 2.83917 2.269354.26245 3.45019 3.65582 3.70207 1.96206 3.26063 4.04582 2.95679 3.847862.75603 2.89654 2.30158 2.29348 3.33728 5.46026 4.09624 32 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 322.98261 4.34033 4.74407 4.16222 2.74015 4.19374 4.48870 1.33722 4.008881.70186 3.16247 4.27271 4.50036 4.14592 4.09048 3.51345 3.21313 1.977554.93581 3.15765 33 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.619580.77255 0.48576 0.95510 33 2.50714 4.52249 3.32761 2.73441 3.994132.87824 3.93263 3.37611 2.83014 3.05077 3.89907 3.20556 3.90546 3.156473.23473 2.09891 1.66153 2.58887 5.34950 4.08041 34 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 34 2.733720.86170 4.21080 3.82223 3.72250 3.51687 4.43487 3.14732 3.50989 2.936753.97687 3.88251 4.17078 3.93344 3.44432 2.99564 3.14856 2.89652 5.251693.70577 35 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.772550.48576 0.95510 35 2.78221 4.93521 3.15849 2.72873 3.66991 3.614313.78500 3.57206 2.57323 3.13265 4.05241 3.20038 4.05162 1.36867 2.921562.93601 3.12692 3.29834 5.09768 2.57435 36 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.019874.32413 5.04648 0.61958 0.77255 0.48576 0.95510 36 2.39232 4.686553.16574 2.46000 3.89951 3.51768 3.74742 2.86223 2.34831 2.93724 3.785113.11031 3.91433 2.90991 3.00803 2.69966 1.85059 2.95170 5.23226 3.9382637 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.485760.95510 37 2.38712 4.95526 2.95139 2.14292 4.22581 3.30071 3.487573.66208 2.41922 3.22834 4.01725 2.96015 3.42523 2.64575 2.85382 2.409152.33462 3.08668 5.43599 3.59495 38 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.324135.04648 0.61958 0.77255 0.48576 0.95510 38 3.32493 5.03100 4.088424.05521 5.14287 0.28143 5.12256 4.87721 4.29611 4.46333 5.45951 4.255164.43330 4.58529 4.45932 3.51325 3.84001 4.31766 6.09414 5.26815 39 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551039 2.77845 5.16916 2.50798 1.90461 4.47239 3.42576 3.73520 3.928932.55535 3.46812 4.26082 2.91770 3.47218 2.74953 3.05304 2.74879 1.689683.54385 5.65411 4.25701 40 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.046480.61958 0.77255 0.48576 0.95510 40 2.55637 3.36322 2.86419 2.494204.07854 3.43147 3.53651 3.49427 2.36324 3.10007 3.91410 2.51497 3.867782.83786 2.95074 2.24490 2.43049 3.00556 5.34651 3.93774 41 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 413.32262 4.56891 5.24679 4.77118 3.84175 4.81012 5.48421 1.14831 4.681432.30177 3.60891 4.94823 5.08094 4.93596 4.85577 4.22682 3.58792 0.994605.88795 4.67682 42 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.619580.77255 0.48576 0.95510 42 2.65825 5.10541 2.72992 2.15627 3.983633.31023 3.53625 3.89170 2.18852 3.23731 4.15424 2.23857 3.82936 2.657482.86654 2.25743 2.79796 3.48165 5.55380 4.15800 43 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 43 2.611824.52680 3.60621 3.51889 4.78008 0.61583 4.68006 4.23876 3.72062 3.952944.83622 3.66170 4.02536 4.00392 3.97409 2.79132 2.85714 3.64017 6.056214.90618 44 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.772550.48576 0.95510 44 2.79049 5.22667 2.25221 2.33027 4.53369 3.279473.53614 4.00930 2.56097 3.52971 4.31450 1.59788 3.89352 2.86397 3.069872.39242 3.03945 3.49407 5.69864 4.13140 45 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.019874.32413 5.04648 0.61958 0.77255 0.48576 0.95510 45 2.49378 3.369322.74714 2.42229 4.10830 3.46961 3.68039 3.52770 2.44255 3.12654 3.936022.51463 3.86384 2.82770 2.94369 2.19290 2.51568 3.13211 5.36588 3.7602446 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.485760.95510 46 2.69732 4.16858 4.15640 3.57668 3.28068 3.80998 4.133661.64485 3.45403 1.84904 3.26899 3.79253 3.96991 3.68559 3.64246 2.817772.62992 2.15853 4.82146 3.52063 47 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.324135.04648 0.61958 0.77255 0.48576 0.95510 47 4.19233 5.38393 4.846054.67977 3.27817 4.29458 4.57157 4.26819 4.41284 3.57367 4.87507 4.748454.85287 4.77414 4.45256 4.39169 4.52083 4.16862 0.32020 3.26075 48 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551048 3.20149 5.67973 0.78816 2.34486 4.76934 3.43076 3.17601 4.558953.07436 4.04716 4.94146 2.92732 4.07402 3.21129 3.65781 3.08955 3.506324.14006 6.03658 4.51417 49 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.046480.61958 0.77255 0.48576 0.95510 49 2.76346 4.98544 3.15831 2.569574.01057 3.55403 3.68400 3.67589 1.47331 2.89158 4.05158 3.07047 3.933822.56322 2.45400 2.78078 2.98070 3.34485 5.41794 3.49426 50 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 502.64257 4.43619 3.56736 3.08202 3.77537 3.52079 4.05811 2.42620 3.009272.79176 3.72810 2.99222 4.03030 3.35753 3.34645 2.85418 1.35329 2.629665.23131 3.98292 51 - - 2.68619 4.42226 2.77521 2.73124 3.46340 2.405143.72496 3.29355 2.67742 2.69356 4.24691 2.90348 2.73741 3.18148 2.898022.37888 2.77521 2.98520 4.58478 3.61472 0.08832 2.54971 5.04648 0.376391.15942 0.48576 0.95510 51 2.52068 4.47414 3.35565 2.74391 3.339683.48613 3.81115 3.00228 2.76056 2.69538 3.20591 3.24034 3.68619 2.824173.12828 2.08694 2.32290 2.59916 4.31681 3.78415 53 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 52 2.778604.87789 1.42239 2.56155 3.13731 3.54366 3.74371 3.49507 2.67769 3.095853.97167 3.06454 3.97169 2.99708 3.12924 2.82286 3.01884 3.20614 4.049283.59798 54 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.772550.48576 0.95510 53 2.94931 5.28328 2.08563 2.39481 4.84632 1.164254.00151 4.36230 3.01231 3.90219 4.74326 2.45548 3.99074 3.18038 3.567292.91382 3.29175 3.89959 6.06470 4.62009 55 - - 2.68621 4.42228 2.775222.73126 3.46357 2.40509 3.72497 3.29357 2.67727 2.69358 4.24693 2.903462.73742 3.18149 2.89804 2.37878 2.77522 2.98521 4.58480 3.61506 0.085092.58845 5.04648 0.73284 0.65497 0.48576 0.95510 54 3.21633 0.354794.79708 4.65548 4.57914 3.72194 5.20787 3.82938 4.49870 3.70850 4.851674.53077 4.45916 4.82584 4.53365 3.48556 3.72159 3.53505 5.86530 4.8563159 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.485760.95510 55 4.07603 5.22780 4.94333 4.71701 1.81696 4.70604 3.644053.73694 4.54415 3.00155 4.31962 4.34626 5.01267 4.44587 4.52415 4.108634.29875 3.67538 3.75731 0.60600 60 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.324135.04648 0.61958 0.77255 0.48576 0.95510 56 3.29932 4.57114 5.139144.65306 3.79025 4.73170 5.36042 1.62171 4.54191 2.21256 3.57888 4.846275.01963 4.80796 4.72647 4.13701 3.56571 0.76183 5.80720 4.58938 61 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.9551057 1.40302 4.31490 3.77731 3.44350 4.52474 3.10962 4.46137 3.906753.45708 3.61908 4.43729 3.53676 3.86941 3.73557 3.76041 1.18441 2.550053.34107 5.86901 4.67381 62 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.046480.61958 0.77255 0.48576 0.95510 58 3.16638 5.52729 0.80512 2.429945.07620 2.30005 4.12607 4.65042 3.22647 4.16769 5.05164 2.99972 4.087593.32219 3.82773 3.09290 3.51563 4.17606 6.23444 4.80691 63 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 593.11990 4.36544 4.32127 3.81191 2.17745 4.09367 3.78203 3.04592 3.238062.64903 3.67717 3.91110 4.44159 3.85035 3.84946 3.39383 3.34718 2.779123.74216 1.06936 64 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.01987 4.32413 5.04648 0.619580.77255 0.48576 0.95510 60 3.51146 4.91649 4.45325 4.08004 2.025674.31163 3.69403 3.46524 3.96389 2.92976 4.07889 4.05659 4.68281 4.075374.10672 2.99801 3.75887 3.31559 3.91189 0.78971 65 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.01987 4.32413 5.04648 0.61958 0.77255 0.48576 0.95510 61 2.725534.31253 4.16671 3.66674 3.67680 3.72642 4.41126 2.29394 3.56150 2.587083.60951 3.86666 4.24179 3.84789 3.80334 2.76599 2.38224 1.10833 5.258334.04936 66 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.02985 4.32413 4.12501 0.61958 0.772550.48576 0.95510 62 2.51216 5.16569 2.33791 2.35923 4.49437 3.193743.66109 3.96480 1.64210 3.46558 4.22781 2.83561 3.85394 2.71011 2.841922.54559 2.88860 3.55105 5.61534 4.21583 67 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.020074.31435 5.03670 0.61958 0.77255 0.49963 0.93333 63 2.49188 4.389553.59551 3.16295 4.09719 3.28177 4.17679 3.38911 3.04040 3.15453 4.028413.43067 3.92288 3.44894 3.44644 2.11805 1.20176 2.68117 5.48692 4.2541168 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 0.02007 4.31435 5.03670 0.61958 0.77255 0.499630.93333 64 1.89508 4.31720 3.70048 3.48388 4.63634 0.95179 4.551594.00807 3.58485 3.73760 4.55934 3.55250 3.87506 3.84242 3.86415 2.565922.50453 3.40204 5.97883 4.79808 69 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.03953 4.314353.67355 0.61958 0.77255 0.49963 0.93333 65 2.63597 3.39258 3.145662.45530 3.91792 3.49097 3.54341 3.31288 2.40023 2.95698 3.79792 3.087853.89316 2.92081 3.00148 1.97899 2.29007 2.96915 5.24220 3.94276 70 - -2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 0.02046 4.29528 5.01763 0.61958 0.77255 0.52575 0.8943266 2.59137 4.97540 2.68695 2.29850 4.05453 3.34590 3.64946 3.593802.42308 3.26160 4.04572 2.57932 3.83844 2.77492 2.90216 1.98570 2.446893.22530 5.46068 4.09334 71 - - 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.02046 4.29528 5.017630.61958 0.77255 0.52575 0.89432 67 2.36239 3.82492 3.07537 2.526454.05085 1.94430 3.77137 3.45853 2.53920 3.08721 3.91418 2.86394 3.874032.94691 3.05153 2.46988 2.65768 3.12923 5.35184 4.04208 72 - - 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 0.02046 4.29528 5.01763 0.61958 0.77255 0.52575 0.89432 682.51473 4.23017 3.73065 3.15738 2.96324 3.66009 3.93184 2.62617 3.083872.43082 2.74387 3.49391 3.33993 3.35931 3.14679 2.64784 2.86016 2.493024.81896 2.22311 73 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 0.02046 4.29528 5.01763 0.619580.77255 0.52575 0.89432 69 2.33893 4.26290 4.21737 3.66773 3.277733.87203 4.32579 2.12233 3.56960 2.41061 3.43058 3.89614 4.28394 3.821333.79298 2.87144 3.03565 1.21453 5.06701 3.86005 74 - - 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.246902.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615030.02293 4.29528 4.70670 0.61958 0.77255 0.52575 0.89432 70 2.495934.60476 3.23381 2.52485 3.79749 3.53767 3.76569 3.14297 2.26081 2.407643.70182 3.15412 3.92810 2.98301 3.03430 2.76728 2.09050 2.55846 5.161153.88294 75 - - 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 0.02051 4.29287 5.01521 0.61958 0.772550.52898 0.88967 71 2.60687 5.12624 2.52016 2.23382 4.44833 2.552083.64005 3.91884 2.12896 3.37075 4.18386 2.86694 3.03933 2.74997 2.895012.33562 2.83629 3.50672 5.58015 4.17955 76 - - 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.020514.29287 5.01521 0.61958 0.77255 0.52898 0.88967 72 2.57816 5.126772.87095 2.24649 4.45454 3.41210 3.40077 3.91086 1.59193 3.41242 4.179092.90547 3.85681 2.60853 2.68264 2.65653 2.67555 3.50960 5.55782 4.1830977 - - 2.68618 4.42225 2.77520 2.73117 3.46354 2.40513 3.72495 3.293542.67741 2.69355 4.24690 2.90347 2.73740 3.18147 2.89801 2.37887 2.775202.98519 4.58477 3.61503 0.09497 3.41028 2.85473 0.52137 0.90068 0.528980.88967 73 3.08258 0.42515 4.66004 4.49479 4.42412 3.61311 5.063513.64868 4.33208 3.53771 4.67608 4.38809 4.34603 4.66309 4.38494 3.352663.58099 3.36315 5.74041 4.70439 79 - - 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.014614.23357 * 0.61958 0.77255 0.00000 * //

Example 12: Determination of DomT Scores

The DomT scores for the LAD domain and the LED domain of the LYSpolypeptides of the invention were determined using the LAD CatalyticDomain HMM from Example 10 and the Lysozyme Enhancing Domain HMM fromExample 11 as described herein are presented in table 5 below.

TABLE 5 DomT scores for LAD and LED domains LAD domain LED domainSequence Amino acid DomT Amino acid DomT numbers score numbers score SEQID NO: 3  84 to 226 202.5 1 to 73 118.3 SEQ ID NO: 6  84 to 226 195.4 1to 73 118.5 SEQ ID NO: 9  81 to 220 199.2 1 to 73 116.7 SEQ ID NO: 12161 to 304 193.4 1 to 72 108.7 76 to 147 104.6 SEQ ID NO: 15  85 to 228200.1 1 to 73 125.2 SEQ ID NO: 18  88 to 230 205.3 1 to 73 119.7 SEQ IDNO: 21  87 to 230 201.4 1 to 73 116.9 SEQ ID NO: 24  90 to 232 201.5 1to 73 119.1 SEQ ID NO: 27  85 to 228 199.8 1 to 73 123 SEQ ID NO: 30  85to 228 202.8 1 to 73 122.6 SEQ ID NO: 33  84 to 226 198.2 1 to 73 115.2SEQ ID NO: 36  83 to 222 194.9 1 to 73 113.1 SEQ ID NO: 39  82 to 225203.0 1 to 72 117.4 SEQ ID NO: 42 161 to 303 192.6 1 to 73 115.8 77 to149 111.2 SEQ ID NO: 45  85 to 227 208.3 1 to 73 124.9 SEQ ID NO: 329  4to 146 205.3 — —

All of the claimed LYS polypeptides have a LAD DomT score of at least170, indicating good homology to the LAD HMM model. Likewise all claimedLYS polypeptides have a LED, had a LED DomT score of at least 100,indicating good homology to the LED HMM model.

Example 13: Activity of LYS Polypeptides as Determined Using ReducingEnds Assay

The LYS polypeptides of the invention were tested according to Example 1at two enzyme concentrations and the results are shown in tables 6 to 8below.

TABLE 6 OD Drop of SEQ ID NO: 3 OD Drop OD Drop LYS polypeptide (5.0μg/ml)¹ (0.7 μg/ml) ¹ SEQ ID NO: 3 5.4 2.4 ¹enzyme concentration

TABLE 7 OD Drop of SEQ ID NO: 6 to 45 OD Drop OD Drop LYS polypeptide(5.0 μg/ml)¹ (0.7 μg/ml) ¹ SEQ ID NO: 6 4.4 2.0 SEQ ID NO: 9 5.2 2.7 SEQID NO: 12 2.4 1.4 SEQ ID NO: 15 6.7 3.2 SEQ ID NO: 21 3.9 2.2 SEQ ID NO:24 3.1 1.8 SEQ ID NO: 27 7.8 4.6 SEQ ID NO: 30 8.7 6.0 SEQ ID NO: 33 8.65.7 SEQ ID NO: 36 5.4 2.9 SEQ ID NO: 39 7.8 4.8 SEQ ID NO: 42 5.1 3.1SEQ ID NO: 45 8.5 3.9 SEQ ID NO: 329 5.0 2.3 ¹enzyme concentration

As can be seen, the LYS polypeptides of the invention display lysozymeactivity as determined using the reducing ends assay.

Example 14: Activity of LYS Polypeptides as Determined Using OD DropAssay

The LYA polyupeptides of the invention were tested according to Example2 at pH4 and the results are shown in tables 8 and 9 below.

TABLE 8 OD Drop against M. luteus OD Drop M. luteus LYS polypeptide 1 h,pH 4 SEQ ID NO: 3 0.116 SEQ ID NO: 6 0.151 SEQ ID NO: 9 0.121 SEQ ID NO:12 0.177 SEQ ID NO: 15 0.125 SEQ ID NO: 21 0.113 SEQ ID NO: 24 0.121 SEQID NO: 27 0.071 SEQ ID NO: 30 0.081 SEQ ID NO: 33 0.052 SEQ ID NO: 360.171 SEQ ID NO: 39 0.154 SEQ ID NO: 42 0.162

TABLE 9 OD Drop against M. luteus OD Drop M. luteus LYS polypeptide 1 h,pH 4 SEQ ID NO: 18 0.078 SEQ ID NO: 329 0.063

As can be seen, the LYS polypeptides of the invention display lysozymeactivity as determined using the traditional OD drop assay against Mluteus.

Example 19: Animal Feed and Animal Feed Additives Comprising a LYSPolypeptide Animal Feed Additive

A formulation comprising the LYS polypeptide of the invention (e.g. SEQID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 or 239)containing 0.01 g to 10 g enzyme protein is added to the followingpremix (per kilo of premix):

5000000 IE Vitamin A 1000000 IE Vitamin D3 13333 mg Vitamin E 1000 mgVitamin K3 750 mg Vitamin B1 2500 mg Vitamin B2 1500 mg Vitamin B6 7666mcg Vitamin B12 12333 mg Niacin 33333 mcg Biotin 300 mg Folic Acid 3000mg Ca-D-Panthothenate 1666 mg Cu 16666 mg Fe 16666 mg Zn 23333 mg Mn 133mg Co 66 mg I 66 mg Se 5.8 % Calcium 25 % Sodium

Animal Feed

This is an example of an animal feed (broiler feed) comprising theanimal feed additive as described above:

62.55% Maize

33.8% Soybean meal (50% crude protein)

1.0% Soybean oil

0.2% DL-Methionine

0.22% DCP (dicalcium phosphate)

0.76% CaCO₃ (calcium carbonate)

0.32% Sand

0.15% NaCl (sodium chloride)

1% of the above Premix

The ingredients are mixed, and the feed is pelleted at the desiredtemperature, e.g. 60, 65, 75, 80, 85, 90 or even 95° C.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1-20. (canceled)
 21. A composition comprising at least 0.01 mg of LYSpolypeptide per kilogram of composition, wherein the polypeptide (a) haslysozyme activity and (b) comprises one or more LAD catalytic domains;wherein the LAD catalytic domain gives a domT score of at least 180 whenqueried using a Profile Hidden Markov Model (HMM) prepared using SEQ IDNOs: 46 to 187 and hmmbuild software program.
 22. The composition ofclaim 21, wherein the polypeptide further comprises one or more lysozymeenhancing domains, wherein the lysozyme enhancing domain gives a domTscore of at least 100 when queried using a Profile Hidden Markov Modelprepared using SEQ ID NOs: 188 to 316 and hmmbuild software program, andwherein the query is carried out using the hmmscan software program. 23.The composition of claim 21, wherein the polypeptide is selected fromthe group consisting of: (a) a polypeptide having at least 80% sequenceidentity to the polypeptide of SEQ ID NO: 3; (b) a polypeptide having atleast 80% sequence identity to the polypeptide of SEQ ID NO: 6; (c) apolypeptide having at least 80% sequence identity to the polypeptide ofSEQ ID NO: 9; (d) a polypeptide having at least 80% sequence identity tothe polypeptide of SEQ ID NO: 12; (e) a polypeptide having at least 80%sequence identity to the polypeptide of SEQ ID NO: 15; (f) a polypeptidehaving at least 80% sequence identity to the polypeptide of SEQ ID NO:18; (g) a polypeptide having at least 80% sequence identity to thepolypeptide of SEQ ID NO: 21; (h) a polypeptide having at least 80%sequence identity to the polypeptide of SEQ ID NO: 24; (i) a polypeptidehaving at least 80% sequence identity to the polypeptide of SEQ ID NO:27; (j) a polypeptide having at least 80% sequence identity to thepolypeptide of SEQ ID NO: 30; (k) a polypeptide having at least 80%sequence identity to the polypeptide of SEQ ID NO: 33; (l) a polypeptidehaving at least 80% sequence identity to the polypeptide of SEQ ID NO:36; (m) a polypeptide having at least 80% sequence identity to thepolypeptide of SEQ ID NO: 39; (n) a polypeptide having at least 80%sequence identity to the polypeptide of SEQ ID NO: 42; (o) a polypeptidehaving at least 80% sequence identity to the polypeptide of SEQ ID NO:45; (p) a variant of the polypeptide selected from the group consistingof SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO:15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ IDNO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 andSEQ ID NO: 45, wherein the variant has lysozyme activity and comprisesone or more amino acid substitutions, and/or one or more amino aciddeletions, and/or one or more amino acid insertions or any combinationthereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 positions; (q) a polypeptidecomprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h),(i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/orC-terminal His-tag and/or HQ-tag; (r) a polypeptide comprising thepolypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k),(l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal extensionof up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aminoacids; and (s) a fragment of the polypeptide of (a), (b), (c), (d), (e),(f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having lysozymeactivity and having at least 90% of the length of the maturepolypeptide.
 24. The composition of claim 21, wherein the LYSpolypeptide comprises one or more motifs selected from the groupconsisting of (a) motif I: (SEQ ID NO: 317) AG[I/L]AT[A/G][I/L][T/V]ES;(b) motif II (SEQ ID NO: 318) V[G/A]XLCQXVQXSAYP; and (c) motif III:(SEQ ID NO: 319) [CGY][YF][VIL][ASTP][DG]X[YF][VIT]X[TS][GAN].


25. The composition of claim 21, wherein the polypeptide is obtained orobtainable from the taxonomic phylum Ascomycota, preferably thetaxonomic subphylum Pezizomycotina.
 26. A granule comprising thecomposition of claim
 21. 27. The granule of claim 26, wherein thegranule is coated.
 28. An animal feed additive comprising thecomposition of claim
 21. 29. An animal feed comprising plant-basedmaterial and the composition of claim
 21. 30. A liquid formulationcomprising the composition of claim 21, wherein the LYS polypeptide isdosed between 0.01% to 25% w/w of the liquid formulation.
 31. The liquidformulation of claim 30, which further comprises 20% to 80% w/w ofpolyol.
 32. A method of improving one or more performance parameters ofan animal comprising administering to the animal the composition ofclaim
 21. 33. A method of preparing an animal feed, comprising mixingthe composition of claim 21 with plant-based material.
 34. An isolatedpolypeptide having lysozyme activity, selected from the group consistingof: (a) a polypeptide having at least 95% sequence identity to thepolypeptide of SEQ ID NO: 3; (b) a polypeptide having at least 94%sequence identity to the polypeptide of SEQ ID NO: 6; (c) a polypeptidehaving at least 80% sequence identity to the polypeptide of SEQ ID NO:9; (d) a polypeptide having at least 80% sequence identity to thepolypeptide of SEQ ID NO: 12; (e) a polypeptide having at least 87%sequence identity to the polypeptide of SEQ ID NO: 15; (f) a polypeptidehaving at least 81% sequence identity to the polypeptide of SEQ ID NO:18; (g) a polypeptide having at least 80% sequence identity to thepolypeptide of SEQ ID NO: 21; (h) a polypeptide having at least 80%sequence identity to the polypeptide of SEQ ID NO: 24; (i) a polypeptidehaving at least 87% sequence identity to the polypeptide of SEQ ID NO:27; (j) a polypeptide having at least 96.2% sequence identity to thepolypeptide of SEQ ID NO: 30; (k) a polypeptide having at least 80%sequence identity to the polypeptide of SEQ ID NO: 33; (l) a polypeptidehaving at least 80% sequence identity to the polypeptide of SEQ ID NO:36; (m) a polypeptide having at least 81% sequence identity to thepolypeptide of SEQ ID NO: 39; (n) a polypeptide having at least 80%sequence identity to the polypeptide of SEQ ID NO: 42; (o) a polypeptidecomprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h),(i), (j), (k), (l), (m), or (n), and a N-terminal and/or C-terminalHis-tag and/or HQ-tag; (p) a polypeptide comprising the polypeptide of(a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), or (n),and a N-terminal and/or C-terminal extension of up to 10 amino acids,e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and (q) a fragment ofthe polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j),(k), (l), (m), or (n), having lysozyme activity and having at least 90%of the length of the mature polypeptide.
 35. The polypeptide of claim34, wherein the polypeptide comprises or consists of amino acids 1 to316 of SEQ ID NO: 2, amino acids 1 to 316 of SEQ ID NO: 3, amino acids 1to 322 of SEQ ID NO: 4, amino acids 1 to 318 of SEQ ID NO: 6, aminoacids 1 to 318 of SEQ ID NO: 7, amino acids 1 to 326 of SEQ ID NO: 8,amino acids 1 to 316 of SEQ ID NO: 10, amino acids 1 to 316 of SEQ IDNO: 11, amino acids 1 to 324 of SEQ ID NO: 12, amino acids 1 to 316 ofSEQ ID NO: 14, amino acids 1 to 316 of SEQ ID NO: 15, amino acids 1 to324 of SEQ ID NO: 16, amino acids 1 to 316 of SEQ ID NO: 18, amino acids1 to 316 of SEQ ID NO: 19, amino acids 1 to 324 of SEQ ID NO: 20, aminoacids 1 to 316 of SEQ ID NO: 22, amino acids 1 to 316 of SEQ ID NO: 23,amino acids 1 to 324 of SEQ ID NO: 24, amino acids 1 to 516 of SEQ IDNO: 26, amino acids 1 to 516 of SEQ ID NO: 27, amino acids 1 to 524 ofSEQ ID NO: 28, amino acids 1 to 317 of SEQ ID NO: 30, amino acids 1 to317 of SEQ ID NO: 31, amino acids 1 to 325 of SEQ ID NO: 32, amino acids1 to 316 of SEQ ID NO: 34, amino acids 1 to 316 of SEQ ID NO: 35, aminoacids 1 to 324 of SEQ ID NO: 36, amino acids 1 to 316 of SEQ ID NO: 38,amino acids 1 to 316 of SEQ ID NO: 39 or amino acids 1 to 324 of SEQ IDNO:
 40. 36. A polynucleotide encoding the polypeptide of claim
 34. 37. Anucleic acid construct or expression vector comprising thepolynucleotide of claim 36 operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.
 38. A recombinant host cell comprising the polynucleotide of claim36 operably linked to one or more control sequences that direct theproduction of the polypeptide.
 39. A method of producing a polypeptidehaving lysozyme activity, comprising: (a) cultivating the recombinanthost cell of claim 38 under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.